CN1550546A - Method for producing L-arginine or L-lysine by fermentation - Google Patents
Method for producing L-arginine or L-lysine by fermentation Download PDFInfo
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- CN1550546A CN1550546A CNA2004100330506A CN200410033050A CN1550546A CN 1550546 A CN1550546 A CN 1550546A CN A2004100330506 A CNA2004100330506 A CN A2004100330506A CN 200410033050 A CN200410033050 A CN 200410033050A CN 1550546 A CN1550546 A CN 1550546A
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01F—MEASURING VOLUME, VOLUME FLOW, MASS FLOW OR LIQUID LEVEL; METERING BY VOLUME
- G01F15/00—Details of, or accessories for, apparatus of groups G01F1/00 - G01F13/00 insofar as such details or appliances are not adapted to particular types of such apparatus
- G01F15/06—Indicating or recording devices
- G01F15/061—Indicating or recording devices for remote indication
- G01F15/063—Indicating or recording devices for remote indication using electrical means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/10—Citrulline; Arginine; Ornithine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01F—MEASURING VOLUME, VOLUME FLOW, MASS FLOW OR LIQUID LEVEL; METERING BY VOLUME
- G01F15/00—Details of, or accessories for, apparatus of groups G01F1/00 - G01F13/00 insofar as such details or appliances are not adapted to particular types of such apparatus
- G01F15/007—Details of, or accessories for, apparatus of groups G01F1/00 - G01F13/00 insofar as such details or appliances are not adapted to particular types of such apparatus comprising means to prevent fraud
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Fluid Mechanics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L-Arginine or L-lysine is produced by culturing a coryneform bacterium having an L-arginine- or L-lysine-producing ability and modified so that glutamine synthetase activity is enhanced, e.g., a coryneform bacterium which is modified so that activity regulation of glutamine synthetase by adenylylation is eliminated, in a medium to produce and accumulate L-arginine or L-lysine in the medium and collecting the L-arginine or L-lysine from the medium.
Description
Technical field
The present invention relates to be specially the method for L-arginine and L-Methionin by using coryneform bacteria to produce L-amino acid.The L-arginine is industrial useful amino acid, can be used as liver function promotor, amino acid transfusion, the composition of amino acid medicament or the like widely, L-Methionin is industrial useful amino acid equally, can be used as animal feedstuff additive, the heath food component, amino acid transfusion etc.
Background technology
To the breeding of microorganism and improvement in being usually used in producing by the L-amino acid of fermentation.In other words because the amino acid whose ability of wild type strain self production L-is very low under many circumstances, event is given auxotrophy or analogue resistance by sudden change or is given metabolism and regulate the method for sudden change or unite these methods of use, these dawns all known in the art.Can obtain the L-amino acid of suitable output according to aforesaid method.But for industrial with low cost production L-amino acid, further improve fermentation yield and definitely be necessary.
It is generally acknowledged that in order to improve fermentation yield, preferable methods is that the enzymic activity in the amino acid biosynthetic pathway is optimized, and promptly strengthens from carbon source and produces amino acid whose biosynthesis system.
Basic aminoacids has extra high nitrogen content.The arginine molecule comprises 6 carbon atoms and 4 nitrogen-atoms, and the Methionin molecule comprises 6 carbon atoms and 2 nitrogen-atoms.
In amino acid fermentation, it is generally acknowledged that the significance level of nitrogen metabolism is not less than the carbon metabolism, in order to improve fermentation yield, the modification of nitrogen metabolism process and modification to the carbon metabolic process are had same importance.In the amino acid bio synthesis system, be to add nitrogen-atoms by being contained in transamination amino in the L-glutamic acid.Therefore, it is generally acknowledged that the concentration that increases interior glutamine of cell and L-glutamic acid can improve amino acid whose fermentation yield.
The disappearance of the odhA gene of coding ketoglurate dehydrogenase has been proved to be the method (WO95/34672) that increases aminoglutaric acid concentration that can be used as, and the glnA gene that strengthens the coding glutamine synthetase has been proved to be the method (European Patent Publication No 1229121) that increases glutamine concentration that can be used as.In addition, now openly the elimination of the adenylylation of glutamine synthetase be a kind of method (European Patent Publication No 1229121) that can effectively strengthen the L-glutaminate supply channel.
But for coryneform bacteria, L-glutaminate or the biosynthetic enhancing of L-L-glutamic acid still are unknown to the influence of L-arginine or L-lysine production ability.
Summary of the invention
One of purpose of the present invention is by modifying coryneform bacteria nitrogen metabolism approach, thereby improves the particularly fermentation yield of L-arginine and L-Methionin of L-amino acid.
Another object of the present invention provides the coryneform bacteria of a kind of L-of having arginine or L-lysine production ability, and described bacillus is modified so that its activity of glutamine synthetase strengthens.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and the activity that described coryneform bacteria has consequently been eliminated the glutamine synthetase that is caused by adenylylation by modification is regulated.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, wherein by the functionally active of following one or more features eliminations by the glutamine synthetase that adenylylation caused:
A) bacterium comprises glutamine synthetase, and the activity adjusting that is caused by adenylylation of described enzyme is eliminated,
B) activity is lowered in the born of the same parents of glutamine synthetase adenylyl transferase,
C) in the proteic born of the same parents of PII activity be lowered and
D) nitrogen metabolism regulate that protein is modified so that the born of the same parents of glutamine synthetase in increased activity.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and wherein the gene of coding glutamine synthetase adenylyl transferase is destroyed on the karyomit(e).
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and it is the amtR gene product that wherein said nitrogen metabolism is regulated albumen, and function causes the activity increase of glutamine synthetase because this gene product can not be brought into normal play.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and wherein the amtR gene on the karyomit(e) is destroyed.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and it is modified so that the arginine repressor function of can not bringing into normal play.
Another object of the present invention provides a kind of above-mentioned coryneform bacteria, and wherein the gene of coding arginine repressor is destroyed on the karyomit(e).
Another object of the present invention provides the method for a kind of L-of production arginine or L-Methionin, and described method comprises:
A) in substratum, cultivate according to each coryneform bacteria in (1) to (8), with produce and accumulation L-arginine or L-Methionin and
B) from this substratum, collect L-arginine or L-Methionin.
According to the present invention, in the process of using coryneform bacteria by fermentative production L-arginine or L-Methionin, thereby the fermentation yield of L-arginine or L-Methionin can be improved by modifying the nitrogen metabolism adjusting.And DNA of the present invention can be used to breed the coryneform bacteria that can produce L-arginine or L-Methionin.
Embodiment
The present inventor has carried out a large amount of research so that achieve the above object.The result; they find; comprising that the glutamine synthetase adenylylation is in the adorned coryneform bacteria of nitrogen metabolism regulation mechanism of major portion; because the increase of glutamine and L-glutamic acid intracellular concentration; showing the amino acid whose ability of superpower fermentative production, particularly is the ability of fermentative production L-arginine and L-Methionin.
Hereinafter, will describe the present invention in detail.
Be used to make up the coryneform bacteria of bacterium of the present invention
Coryneform bacteria comprises and is divided into brevibacterium sp, but unified at present those (Int.J.Syst.Bacteriol., 41,255 (1981)) that are called corynebacterium, and comprise the bacterium that belongs to the closely-related brevibacterium sp of corynebacterium.Coryneform case introduction like this is as follows:
Have a liking for etheric acid coryneform bacteria (Corynebacterium acetoacidophilum)
Vinegar paddy coryneform bacteria (Corynebacterium acetoglutamicum)
Alkanolyticum coryneform bacteria (Corynebacterium alkanolyticum)
Corynebacterium callunae (Corynebacterium callunae)
Corynebacterium glutamicum (Corynebacterium glutamicum)
Flower of Greenish Lily coryneform bacteria (Corynebacterium lilium)
Melassecola coryneform bacteria (Corynebacterium melassecola)
Thermoaminogenes coryneform bacteria (Corynebacterium thermoaminogenes)
Man of great strength's coryneform bacteria (Corynebacterium herculis)
Extension brevibacterium (Brevibacterium divaricatum)
Brevibacterium flavum (Brevibacterium flavum)
Immariophilum tyrothricin (Brevibacterium immariophilum)
Brevibacterium (Brevibacterium lactofermentum)
Rose-colored tyrothricin (Brevibacterium roseum)
Brevibacterium saccharolyticum (Brevibacterium saccharolyticum)
Give birth to sulphur tyrothricin (Brevibacterium thiogenitalis)
Brevibacterium ammoniagenes (Brevibacterium ammoniagenes)
Brevibacterium albus (Brevibacterium album)
Brevibacterium cerinus (Brevibacterium cerinum)
Have a liking for ammonia microbacterium (Microbacterium ammoniaphilum)
Particularly, exemplified the bacterial strain of these bacteriums below
Have a liking for etheric acid corynebacteria A TCC13870
Vinegar paddy corynebacteria A TCC15806
Alkanolyticum corynebacteria A TCC21511
Corynebacterium callunae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Flower of Greenish Lily corynebacteria A TCC15990
Melassecola corynebacteria A TCC17965
Efficiens coryneform bacteria (Corynebacterium efficiens) AJ12340 (FERM BP-1539)
Man of great strength's corynebacteria A TCC13868
Extension brevibacterium ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Immariophilum tyrothricin ATCC14068
Brevibacterium ATCC13869
Rose-colored tyrothricin ATCC13825
Brevibacterium saccharolyticum ATCC14066
Give birth to sulphur tyrothricin ATCC19240
Brevibacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium albus ATCC15111
Brevibacterium cerinus ATCC15112
Have a liking for ammonia microbacterium ATCC15354
These bacterial strains can be available from as American type culture collection (U.S., VA 20110-2209, Manassas, Boulevard university 10801).In other words, each bacterial strain has its specified preserving number, can ask to provide these bacterial strains according to the preserving number of each bacterial strain.In the catalogue of American type culture collection, indicated preserving number corresponding to each bacterial strain.
In addition, the AJ12340 bacterial strain is preserved in Industrial Technology Institute life engineering Industrial Technology Research Institute of Ministry of International Trade and Industry on October 27th, 1987, independent administration mechanism, Independent Administrative Leged Industrial Technology Complex Inst, biological (the Chou Dai-6 of depositary institution of international monopoly, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postcode: 305-5466)) as the international preservation under the budapest treaty, preserving number is FERM BP-1539.The AJ12418 bacterial strain is preserved in Industrial Technology Institute life engineering Industrial Technology Research Institute of Ministry of International Trade and Industry as the international preservation under the budapest treaty on January 5th, 1989, and preserving number is FERMBP-2205.
Among the present invention, term " produce L-arginic ability " is meant when cultivating bacterial strain of the present invention in substratum, the arginic ability of accumulation L-in substratum.The arginic ability of this production L-can be coryneform bacteria wild strain self natural characteristics, also can be to be endowed or the enhanced characteristic by breeding.
Among the present invention, the term ability of Methionin " produce L-" is meant when cultivating bacterial strain of the present invention in substratum, the ability of accumulation L-Methionin in substratum.The ability of this production L-Methionin can be coryneform bacteria wild strain self natural characteristics, also can be to be endowed or the enhanced characteristic by breeding.
Having the coryneform bacteria of producing L-Methionin or the arginic ability of L-can obtain by giving coryneform bacteria wild type strain production L-Methionin or the arginic ability of L-.Be used to breed coryneform bacteria, the ordinary method of Escherichia etc. can be used to give it and produce L-Methionin or the arginic ability of L-.For example, described method comprises, but be not limited to obtain auxotrophic mutant, mutant strain is regulated in analogue resistant strain or metabolism, generates recombinant strain, and the arginic biosynthesis system enzyme of L-Methionin or L-is strengthened (referring to " amino acid fermentation " in the described recombinant strain, the Japan Scientific Societies Press[Gakkai Shuppan center], the 1st edition, on May 30th, 1986 published, the 77-100 page or leaf) etc.Breeding when having the bacterial strain of producing L-Methionin or L-arginine ability, auxotroph, analogue resistance, metabolism regulate that characteristic such as sudden change can be given separately or two or give multinomial the associating.Described biosynthesis system enzyme can strengthen separately or two or morely unite enhancing.In addition, giving of characteristics such as auxotroph, analogue resistance, metabolism adjusting sudden change can be united use with enhancing biosynthesis system enzyme.
Coryneform bacteria with L-arginine throughput is not circumscribed, as long as they have the arginic ability of the L-of production.Described coryneform example includes, but are not limited to the coryneform bacteria of anti-particular agent, and described medicament for example is a sulfa drugs, 2-thiazole L-Ala, pantonine hydroxypentanoic acid etc.; Except that the thiazole of anti-2-L-Ala, or L-Histidine, L-proline(Pro), L-Threonine, L-Isoleucine, L-methionine(Met) or the auxotrophic coryneform bacteria of L-tryptophane (Japanese patent laid-open No.54-44096); The coryneform bacteria (Japanese patent laid-open No.57-18989) of the propanedioic acid of anti-ketone, fluorine propanedioic acid or single gifblaar poison, the coryneform bacteria of anti-smart ammonia alcohol (Japanese patent laid-open No.62-24075); The coryneform bacteria of the guanidine of anti-X-(X represents the derivative of lipid acid or aliphatic chain, Japanese patent laid-open No.2-186995).
Can produce the arginic coryneform bacteria of L-can be bred and be anti-5-AzU, 6-azauracil, 2-deracil, 5 FU 5 fluorouracil, 5-bromouracil, U-18496,6-U-18496 etc.; It can be bred is hydroxamic acid of anti-arginine the (arginine hydroxamate) and the deracil of anti-2-; Can be bred and be hydroxamic acid of anti-arginine the (arginine hydroxamate) and the azauracil of anti-6-(referring to Japanese patent laid-open No.49-126819); Can be bred and be analogue of anti-Histidine the or tryptophane analogue (referring to Japanese patent laid-open No.52-114092); Can be bred and be auxotroph (referring to Japanese patent laid-open No.52-99289) at least a in methionine(Met), Histidine, Threonine, proline(Pro), Isoleucine, Methionin, VITAMIN B4, guanine and the uridylic (or uridylic precursor); Can be bred and be the hydroxamic acid of anti-arginine the (arginine hydroxamate) (referring to Japanese patent application publication No. 51-6754); Can be bred and be succsinic acid auxotroph or the analogue of anti-nucleic acid base the (referring to Japanese patent laid-open No.58-9692); Can be bred for metabolism arginine anergy with by being bred and be antagonist of anti-arginine the and anti-canavanine, and be bred and be Methionin auxotroph (referring to Japanese patent laid-open No.52-8729); Can be bred and be anti-arginine, arginine hydroxamic acid (arginine hydroxamate), homoarginine, D-arginine and canavanine, perhaps be bred and be hydroxamic acid of anti-arginine the (arginine hydroxamate) and 6-azauracil (referring to Japanese patent laid-open No.53-143288); Can be bred and be anti-canavanine (referring to Japanese patent laid-open No.53-3586) or the like.
Can generate the arginic coryneform specific examples of L-and comprise following bacterial strain:
Brevibacterium flavum AJ11169 (FERM P-4161)
Brevibacterium AJ12092 (FERM P-7273)
Brevibacterium flavum AJ11336 (FERM P-4939)
Brevibacterium flavum AJ11345 (FERM P-4948)
Brevibacterium AJ12430 (FERM BP-2228)
AJ11169 strain and AJ12092 strain are the strain of the thiazole of anti-2-L-Ala, and it is disclosed among the Japanese patent laid-open No.54-44096; The AJ11336 strain is the bacterial strain of the pure and mild anti-Sulphadiazine Sodium of anti-smart ammonia, and it is disclosed in open (KOKOKU) number 62-24075 of Japanese Patent; The AJ11345 strain is the bacterial strain with anti-smart ammonia alcohol, 2-thiazole L-Ala, Sulphaguanidine and histidine auxotroph, and it is disclosed in Japanese patent application publication No. 62-24075; The AJ12430 strain is the bacterial strain of octyl group guanidine (octylguanidine) and the thiazole of anti-2-L-Ala, and it is disclosed among the Japanese patent laid-open No.2-186995.
The AJ11169 bacterial strain is preserved in Industrial Technology Institute life engineering industrial research institute of Ministry of International Trade and Industry on August 3rd, 1977, independent administration mechanism, Independent Administrative Leged Industrial Technology Complex Inst, biological (the Chou Dai-6 of depositary institution of international monopoly, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postcode: 305-5466)) preserving number is FERM P-4161.Subsequently, according to budapest treaty, transfer international preservation on September 27th, 1999, preserving number is FERM BP-6892.
The AJ12092 bacterial strain is preserved in Industrial Technology Institute life engineering industrial research institute of Ministry of International Trade and Industry September 29 nineteen eighty-three, and preserving number is FERM P-7273.Subsequently, according to budapest treaty, transfer international preservation on October 1st, 1999, preserving number is FERM BP-6906.
The AJ11336 bacterial strain is preserved in Industrial Technology Institute life engineering industrial research institute of Ministry of International Trade and Industry on April 25th, 1979, and preserving number is FERM P-4939.Subsequently, according to budapest treaty, transfer international preservation on September 27th, 1999, preserving number is FERM BP-6893.
The AJ11345 bacterial strain is preserved in Industrial Technology Institute life engineering industrial research institute of Ministry of International Trade and Industry on April 25th, 1979, and preserving number is FERM P-4948.Subsequently, according to budapest treaty, transfer international preservation on September 27th, 1999, preserving number is FERM BP-6894.
The AJ12430 bacterial strain on December 26th, 1988 be preserved in Ministry of International Trade and Industry's Industrial Technology Institute life engineering industrial research as the international preservation under the budapest treaty, preserving number is FERM BP-2228.
Can in breeding, give or strengthen the throughput of L-Methionin by the method for following introducing sudden change.Described artificial mutant such as following: the mutant of anti-S-(2-amino-ethyl)-halfcystine (hereinafter be called " AEC "); The mutant of some amino acid of growth needs such as homoserine (referring to Japanese patent application publication No. 4828078 and 566499); Anti-AEC and needs are as amino acid whose mutant (referring to U.S. Patent Publication No. 3,708,395 and 3,825,472) such as L-leucine, L-homoserine, L-proline(Pro), L-Serine, L-arginine, L-L-Ala, L-Xie Ansuans; The amino hexanolactam of anti-DL-, amino lauryl lactam, aspartic acid analogue, sulfonamide, quinone and N-dodecyl be leucic can to generate the mutant that can generate L-Methionin of the mutant of L-Methionin and anti-oxaloacetic decarboxylase or respiratory system enzyme inhibitors (referring to Japanese patent laid-open No.5053588,5031093,52102498,539394,5386089,559783,559759,5632995,5639778, Japanese patent application publication No. 5343591,531833); The mutant that can generate L-Methionin (referring to Japanese patent laid-open 559784,568692) that needs inositol or acetate; To the fluorine pyruvic acid or to the mutant that can generate L-Methionin (referring to Japanese patent laid-open No.5386090) of 34 ℃ or higher temperature sensitivity; The mutant that can generate L-Methionin (referring to U.S. Patent Publication No. 4,411,997) of ethylene glycol resistant that belongs to the bacterium of brevibacterium sp or corynebacterium.
To illustrate below by strengthening L-arginine or L-Methionin biosynthesis system enzymic activity and give or strengthen the method for L-amino acid generative capacity.
Zymosthenic realization can suddenly change by introducing in the gene of codase, thereby the interior activity of born of the same parents of this enzyme is strengthened, or by using the gene recombination technology of using this gene.
Enzyme in the L-arginine biosynthetic pathway can be to be selected from the following enzyme one or more: N-acetyl glutamy phosphoric acid reduction enzyme (argC), ornithine acetyltransferase (argJ), N-acetylglutamat kinases (argB), acetyl-ornithine transaminase (argD), ornithine carbamyl transferase (argF), argininosuccinate synthetase (argG) and argininosuccinate lyase (argH), carbamyl phosphate synthetase (carAB).The unnamed gene of encoding such enzymes provides in the parenthesis of each enzyme back respectively.
Can strengthen by the active sudden change example of above-mentioned enzyme encoded protein and comprise promoter sequence sudden change, or the genes encoding region mutation etc., the former increases gene transcription quantity, and the latter increases the specific activity of zymoprotein etc.
And, strengthening enzymic activity for the applying gene recombinant technology, can realize by following method, for example increase the copy number of the gene of codase in the cell.For example, can be connected with the carrier (carrier of preferred multiple copied type) of functionating in microorganism by the dna fragmentation that will contain this gene and prepare recombinant DNA, be used to transform microorganism then.
Except aforementioned method based on gene amplification, reinforcing gene expression can also realize by the following method, as being stronger promotor (WO00/18935) with expression of gene regulating and controlling sequence on chromosomal DNA or the plasmid such as promoter replacement.The example of strong promoter comprises lac promotor, trp promotor, trc promotor etc.And, can also make its modification become stronger promotor the promoter region of introducing lysE genes such as Nucleotide replacement.Promotor by such replacement or modify can enhancing gene expression.The modification of this expression regulation sequence can be carried out jointly with the copy number that increases gene.
The example of the gene of L-Methionin biosynthesis system comprises, for example, the gene of coding E.C. 2.7.2.4. α-protein subunit or β-protein subunit, described subunit through the collaborative feedback inhibition of L-Methionin and L-Threonine by desensitization (International Patent Application WO 94/25605), from coryneform wild-type phosphoenolpyruvate carboxykinase gene (Japanese patent laid-open No.60-87788), the gene (Japanese patent application publication No. 6-55149) from coryneform wild-type dihydrodipicolinic acid synthase or the like of encoding.
The method of aforementioned enhancing L-arginine biosynthesis system enzymic activity can be equally applicable to L-Methionin.
In addition, can make arginine repressor in the bacterial cell function of can not bringing into normal play, thereby give or strengthen L-Methionin or the arginic throughput of L-by modifying coryneform bacteria.
The arginine biosynthesis system gene for example expression of N-acetylglutamide synthase gene can be subjected to arginic obvious inhibition in the substratum usually.Coryneform bacteria was shown already the generation of some enzymes is subjected to the arginic inhibition of L-in the L-arginine biosynthetic pathway.And, there is report to point out, though some enzymes in the L-arginine biosynthetic pathway can be subjected to the arginic inhibition of L-, but the L-arginine does not exist in some coryneform bacteria mutant strains the restraining effect of these enzymes, and these bacteriums also show the increase (Agric.Biol.Chem. of L-arginine accumulation volume, 43 (1), 105,1979).
In addition, for intestinal bacteria, the gene of the repressor of L-arginine biosynthesis system (arginine repressor) and this repressor of encoding was identified (the Proc.Natl.Acad.Sci. U.S., (1987) already, 84 (19) 6697-701), and the interaction of repressor albumen and multiple L-arginine biosynthetic pathway gene (Proc.Natl.Acad.Sci., the U.S., (1987) have been carried out studying, 84 (19) 6697-701, J.Mol.Biol. (1992), 226,367-386).
Arginine be by intermediate such as ornithine and citrulline via the special biosynthetic pathway of arginine synthetic, and carbamyl phosphate also participates in this approach.Therefore, in order to increase the route of synthesis that arginic fermentation yield just is necessary to strengthen carbamyl phosphate.
Carbamyl phosphate is to use carbonate ion, and glutamine and ATP produce.In coryneform bacteria, carbonate ion is from nutrient solution, and ATP generates in the carbohydrate metabolism process.Therefore, the supply of glutamine is very important to the generation of carbamyl phosphate.
Based on foregoing, thereby consider that L-Methionin and the arginic throughput of L-can be by making that can not the bring into normal play modification of function and the method Joint Implementation of following increase activity of glutamine synthetase of arginine repressor further strengthened.
The present invention's " arginine repressor " is meant to have the protein that suppresses L-arginine biosynthesizing effect, if this proteinic genetic expression quantity of coding increases in the microorganism, L-arginine throughput promptly descends, if this genetic expression quantity descends or this albumen disappears, L-arginine throughput promptly improves.Hereinafter, the gene of coding arginine repressor is called the argR gene again.Term " arginine repressor can not bring into normal play function " is meant with wild type strain or unmodified bacterial strain and compares that the activity of arginine repressor reduces or disappears.
The nucleotide sequence of the argR gene of brevibacterium flavum and its amino acid sequence coded are as shown in SEQ IDNO:15, and aminoacid sequence is as shown in SEQ ID NO:16.
In the present invention, the arginine repressor is in order to reduce its active purpose, and it can have the aminoacid sequence that the one or more sites that are included in above-mentioned arginine repressor aminoacid sequence lacked, replace, insert or added one or more amino-acid residues.
Position and the amino acid whose type of amino-acid residue in protein three-dimensional structure depended in the variation of " one or more " total number of atnino acid purpose.This is because following reason.In other words, some amino acid have high homology each other, and the difference between these amino acid can the proteinic three-dimensional structure of remarkably influenced.Therefore, arginine repressor mutant of the present invention can be to have 30-50% or higher with whole amino-acid residues of forming the arginine repressor, preferred 50-70% or higher, more preferably 80-90% or higher, most preferably 95% or higher homology and have the active protein of arginine repressor.
The protein DNA substantially the same with aforementioned arginine repressor of encoding is included under the stringent condition can have the active DNA of arginine repressor with argR gene recombination and its encoded protein matter." stringent condition " is meant under this condition, forms so-called specific hybrid body, and do not form the non-specific hybridization body.Use any numerical value and know that it all is very difficult illustrating this condition.But, for example, stringent condition comprises such condition, under this condition, has the DNA of high homology, for example 50% or higher homology, preferred 70% or higher homology, more preferably 80% or higher homology, most preferably 90% or the DNA of higher homology can hybridize each other, can not hybridize each other but homology is lower than the DNA of above-mentioned level.Perhaps, stringent condition comprises such condition: under this condition, DNA under representative condition corresponding to Southern hybridization, i.e. 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1% SDS can be hybridized under 60 ℃ each other.
The example that reduces coryneform arginine repressor activity methods comprises: for example with mutagenic compound commonly used in uviolizing or the mutagenic treatment such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid processing coryneform bacteria, pick out then and show as the active mutant strain that reduces of arginine repressor.Except sudden change is handled, showing as the active coryneform bacteria that reduces of arginine repressor can also be available from following method: the partial sequence by disappearance argR gene makes the argR gene of the coding arginine repressor after modifying can't generate the arginine repressor of normal function, transform coryneform bacteria with the DNA that contains this argR gene (absence type argR gene), thereby make argR gene on the reorganization destruction karyomit(e) takes place between the argR gene on above-mentioned absence type argR gene and the karyomit(e).
The gene disruption of glnE gene described later can adopt about the same manner of the gene disruption of argR gene and implement.
The source of argR gene is not particularly limited, as long as it has homology to a certain degree, can carry out homologous recombination with the argR gene of target microorganism.Specifically, coryneform argR gene includes, but not limited to argR gene of above-mentioned brevibacterium flavum and the argR gene of Corynebacterium glutamicum (GenBank numbers AF049897).These argR gene height homologies even it is believed that the coryneform argR gene that belongs to different genera with the coryneform bacteria of desiring to carry out the argR gene disruption, also can be used for the destruction of this gene.
Make up coryneform bacteria of the present invention
Coryneform bacteria of the present invention produces ability for having above-mentioned L-Methionin or L-arginine, and is modified so that the coryneform bacteria of glutamine synthetase (being also referred to as " GS ") increased activity in the born of the same parents.
In coryneform bacteria of the present invention is bred, can at first implement giving of L-Methionin or L-arginine throughput, also can at first implement the active enhancing of GS.
Term " modify so that born of the same parents in GS increased activity " is meant for example wild-type coryneform bacteria of bacterial strain that the GS activity of each cell is higher than non-modification.For example, can refer to those situations of the quantity increase of GS molecule in each cell, those situations of active increase of GS or the like in each GS molecule." GS activity " is meant that catalysis is from the ability of the reaction of L-glutamic acid and ammonia generation glutamine by using ATP.In addition, the wild-type coryneform bacteria of purpose use can be as a comparison, as brevibacterium ATCC13869.
Concrete, the coryneform bacteria of preferred GS increased activity, for example the GS activity shows as 100-150nmol/min/mg intracellular protein or higher coryneform bacteria, or the GS activity is higher than wild strain 2-3 coryneform bacteria doubly.But coryneform bacteria of the present invention is not limited thereto.The GS activity can be with being disclosed in Journal ofFermentation and Bioengineering, Vol.70, and No.3,182-184, the method in 1990 is measured.Intracellular protein can be used in addition, carries out quantitatively as the albumen test (Bio-Rad) of adopting the bovine serum albumin standard substance.
By realizing that as the genetic expression that strengthens coding GS activity of glutamine synthetase strengthens in the born of the same parents.Can obtain the increase of genetic expression quantity by the copy number that increases coding GS gene.For example by the gene fragment of GS and the carrier that works in bacterium of will encoding, preferably multiple copied type carrier connects the preparation recombinant DNA and is used to transform coryneform bacteria.
From coryneform bacteria or from any GS gene of other organism such as Escherichia bacterium all is operable.Wherein, owing to be easy to express preferably from coryneform gene.
Gene glnA (FEMS Microbiology Letters 81-88 (154), 1997) and gln42 (Japanese patent laid-open No.2002-300887, EP1229121, Microbiology Letters such as L.Nolden, 201, (2001) 91-98) has been reported as the gene of coding coryneform bacteria GS.
The aminoacid sequence of the nucleotide sequence of gene glnA and this genes encoding of brevibacterium is shown among the SEQ ID NO:19, and aminoacid sequence is shown among the SEQ ID NO:20.Although disclosed initiator codon in SEQ ID Nos:19 and 20 (Nucleotide 1-3) amino acids coding is a Xie Ansuan, it very may be methionine(Met).The nucleotide sequence of gene glnA2 and amino acid sequence coded thereof are disclosed among Japanese patent laid-open No.2002-300887 and the EP1229121.
Among the present invention, GS can be included in one or more sites and lack, replaces, inserts or add one or more amino-acid residues, as long as the GS activity does not lower.The number change of amino-acid residue " two or more " depends on according to position and the amino acid whose type of amino-acid residue in proteinic three-dimensional structure.But the mutant of GS of the present invention can be to have 30-50% or higher with whole amino-acid residues of forming GS, preferred 50-70% or higher, more preferably 80-90% or higher, most preferably 95% or higher homology and have the active protein of GS.
The protein DNA substantially the same with aforementioned GS of encoding is included under the stringent condition and can has the active DNA of GS with glnA or glnA2 gene recombination and encoded protein matter.Stringent condition comprises such condition: under this condition, have high homology and for example have 50% or higher homology, preferred 70% or higher homology, more preferably 80% or higher homology, most preferably 90% or the DNA phase mutual cross of higher homology, but the DNA that is lower than above-mentioned homology is not hybridized mutually.Perhaps, stringent condition comprises such condition: under this condition, DNA under typical wash conditions corresponding to Southern hybridization, i.e. 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS, 60 ℃ of hybridization down.
In addition, thus available modification bacterium and reduce or eliminate the glutamine synthetase adenylylation and make that activity of glutamine synthetase strengthens in the coryneform born of the same parents.
By the tyrosine residues in the adenylylation aminoacid sequence, change GS into inactivation form (Proc.Natl.Acad.Sci., the U.S., 642-649, (58) 1967; J.Biol.Chem., 3769-3771, (243) 1968).So can strengthen intracellular GS activity by eliminating the GS adenylylation to its active control.Reducing or eliminating of adenylylation used herein not only refers to eliminate fully basically adenylylation, and refers to reduce such adenylylation, thereby makes the increased activity of GS in the cell." minimizing " is meant that the adenylylation of comparing GS with the corynebacterium strain of wild-type corynebacterium strain or unmodified reduces.The wild-type coryneform bacteria of purpose use can be as a comparison, as brevibacterium ATCC13869.
Hereinafter will illustrate and reduce the method for eliminating GS adenylylation activity control.
The modification in glutamine synthetase adenylylation site
In intestinal bacteria etc., the adenylylation of GS can utilize glutamine synthetase adenylyl transferase (hereinafter being called ATase, Proc.Natl.Acad.Sci., the U.S., 1703-1710 page or leaf, (58), 1967) usually.In coryneform bacteria, GS changes inactive form (FEMS Microbiology Letters, 303-310, (173), 1999 into by the adenylylation of glutamine synthetase adenylyl transferase; FEMSMicrobiology Letters, 201, (2001), 91-98).There is report to point out, in coryneform bacteria, 405 tyrosine residues generation adenylylations (FEMS Microbiology Letters, 303-310, (173), 1999) of the glnA gene product shown in the sequence of Genebank registration number Y13221.The caused GS inactivation of this adenylylation can be eliminated by following method: make tyrosine residues by another amino-acid residue thereby import sudden change in the glnA gene, replace as phenylalanine residue.
In addition, the same activity control that all can be subjected to adenylylation of aforementioned glnA2 gene product with the glnA gene product.Therefore; the inactivation that adenylylation caused by the GS of glnA2 coded by said gene just can be eliminated by the following method, promptly introduces sudden change so that 405 corresponding amino-acid residues of tyrosine residues are substituted by another amino-acid residue such as phenylalanine in the glnA2 gene.
The DNA that coding has been eliminated the GS of activity control due to the adenylylation can obtain by the following method, promptly modifies glnA or glnA2 sequence and makes this sequence comprise sudden change, and this sudden change can be eliminated the activity control of adenylylation to coded GS.The mutator gene that obtains can adopt any method similar to the employed method of aforementioned enhancing L-arginine biosynthesis system gene and be introduced into coryneform bacteria.
Except eliminating sudden change by activity control due to the adenylylation; the aminoacid sequence of the adorned GS in adenylylation site also can be included on one or more sites and lack, replaces, inserts or add one or more amino-acid residues, as long as the GS activity does not lower.
The active attenuating of glutamine synthetase adenylyl transferase
The activity control of GS can also realize by the activity that lowers glutamine synthetase adenylyl transferase (ATase) in the born of the same parents due to the elimination adenylylation.The activity that lowers ATase not only refers to eliminate fully its activity here but also refers to that following activity lowers, and promptly compares active attenuating of ATase in the born of the same parents with coryneform wild strain or non-modification strain.For example, those situations that the number of copies of ATase molecule reduces in each cell, those situations of each ATase molecule ATase specific activity attenuating or the like.The wild-type coryneform bacteria of purpose use can be as a comparison, as brevibacterium ATCC13869.
The gene glnE of brevibacterium ATCC 13869 strains has been illustrated the gene (EP1229121) for coding ATase.The nucleotide sequence of this gene and amino acid sequence coded thereof are shown among the SEQ ID NO:17, and aminoacid sequence is shown among the SEQ ID NO:18.
In the present invention, in order to reduce its active purpose, one or more sites that ATase can have an aminoacid sequence that is included in above-mentioned ATase lack, replace, insert or add the aminoacid sequence of one or more amino-acid residues.
The number change of amino-acid residue " two or more " depends on position and the amino acid whose type of amino-acid residue in proteinic three-dimensional structure.But the mutant of ATase of the present invention can be to have 30-50% or higher with whole amino-acid residues of forming ATase, preferred 50-70% or higher, more preferably 80-90% or higher, most preferably 95% or higher homology and have the active protein of ATase.
The protein DNA substantially the same with aforementioned ATase of encoding is included under the stringent condition and glnE gene recombination and encoded protein matter have the active DNA of ATase." stringent condition " comprise such condition, under this condition, DNA with high homology, for example 50% or higher homology, preferred 70% or higher homology, more preferably 80% or higher homology, most preferably 90% or the DNA of higher homology can hybridize each other, can not hybridize each other but homology is lower than the DNA of above-mentioned level.Perhaps, stringent condition comprises such condition: under this condition, DNA under typical wash conditions corresponding to Southem hybridization, i.e. 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS can be hybridized under 60 ℃ each other.
The example that reduces coryneform ATase activity methods comprises: for example with mutagenic compound commonly used in uviolizing or the mutagenic treatment such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid processing coryneform bacteria, pick out then and show as the active mutant strain that reduces of ATase.Except sudden change is handled, showing as the active coryneform bacteria that reduces of ATase can also be available from following method: the partial sequence by disappearance glnE gene makes the ATase encoding gene (glnE) after modifying can't generate the ATase of normal function, transform coryneform bacteria with the DNA that contains this glnE gene (absence type glnE gene), thereby make glnE gene on the reorganization destruction karyomit(e) takes place between the glnE gene on above-mentioned absence type glnE gene and the karyomit(e).Carry out the gene disruption that gene replaces by homologous recombination and be established, wherein utilize linear DNA, contain the method for the plasmid or the like of temperature sensitive replication orgin.
For example can make glnE gene on the host chromosome by absence type glnE gene substitution with following method.That is, there is the marker gene of resistance to prepare recombinant DNA, transforms coryneform bacteria with recombinant DNA then by the glnE gene of insertion temperature sensitivity replication orgin, sudden change with to medicine such as paraxin.In addition, under the temperature that the temperature sensitive replication orgin does not play a role, cultivate the transformant bacterial strain that obtains, in containing the substratum of medicine, cultivate the transformant bacterial strain then, obtain mixing in the chromosomal DNA transformant bacterial strain of recombinant DNA.
Recombinant DNA is being mixed in the bacterial strain of chromosomal DNA with aforesaid method, the glnE gene and the original glnE gene recombination that is present on the karyomit(e) of sudden change, and two fusion genes of karyomit(e) glnE gene and absence type glnE gene insert in the karyomit(e), thereby the other parts of recombinant DNA (carrier segments, temperature sensitive replication orgin and drug resistance mark) promptly are present between two fusion genes.
In being connected to the glnE of plasmid, use the internal sequence that does not comprise any promotor and initiator codon.Therefore, the structure gene of GlnE is interrupted by such structure that the other parts of recombinant DNA (carrier segments, temperature sensitive replication orgin and drug resistance mark) are present between two fusion genes, so GlnE loses function.
In addition, the glnE that has lacked internal sequence can be used as absence type glnE equally and is used.In this case, when having inserted two fusion genes of karyomit(e) glnE and absence type glnE in the chromosomal DNA, natural glnE gene has comparative advantage, thereby the conversion bacterial strain can be expressed normal ATase.Like this, in order on chromosomal DNA, only to keep absence type glnE gene, taked the method for a copy glnE gene being removed from chromosomal DNA together with carrier segments (comprising temperature sensitive replication orgin and drug resistance marker) by two glnE gene recombination.In this case, allow natural glnE gene be retained on the chromosomal DNA, and absence type glnE gene is excised from karyomit(e), perhaps opposite, allow absence type glnE gene be retained on the chromosomal DNA, and natural glnE gene is excised from karyomit(e).In both cases, when under the temperature that can work at temperature sensitive replication orgin during culturing cell, cut DNA is retained in the cell as plasmid.Subsequently, if in the temperature of temperature sensitive replication orgin unable to get up effect during culturing cell, the glnE gene on this plasmid can be eliminated from cell together with plasmid.Like this, can select to keep on a kind of karyomit(e) the bacterial strain of absence type glnE gene by PCR, Southern hybridization or similar method, thereby obtain the ruined bacterial strain of glnE gene.
Perhaps, can use and in coryneform bacteria, to replace the responsive plasmid of said temperature to carry out gene disruption by the plasmid of self-replicating.Can not in coryneform bacteria, comprise pHSG299 (TaksraShuzo) and pHSG399 (Taksra Shuzo) etc. by the example of the plasmid of self-replicating.
Bacterial strain with ruined glnE can obtain with aforesaid method.
The source that is used for the glnE gene of gene disruption does not have concrete restriction, as long as the homology degree between the original glnE gene that comprises is enough to guarantee can take place between the two reorganization in this glnE gene and the coryneform bacteria.For example, the ATases (the Genbank registration number is Y17736) of ATases of mycobacterium tuberculosis (Mycobacterium tuberculosis) (the Genbank registration number is Z70692) and streptomyces coelicolor (Streptomyces coelicolor) and the homology between the coryneform Atase are respectively 51.9% and 33.4%.(see Japanese patent laid-open No.2002-300887, EP1229121)
Reduce the PII protein-active
Also can be by reducing the active adenylylation inactivation that weakens GS in the proteic born of the same parents of PII.Known PII albumen has also participated in the adenylylation of ATase to GS.PII albumen is the active signal transfer protein of a kind of control GS, it is reported that it can be by Uridyl transferase (UTase) uridine acidylate.The PH albumen of uridine acidylate can promote the go adenylylation of ATase to GS, and goes the PII albumen of uridine acidylate can promote the adenylylation of ATase to GS.
Report in addition, in UTase defective bacterial strain, GS height adenylylation (J.Bacteriology, 569-577, (134) 1978).The phenotype of this excessive adenylylation can be suppressed (J.Bacteriology, 816-822, (164) 1985) by the proteic sudden change of PII.That is to say that the adenylylation inactivation of GS also can weaken by reducing the PII protein-active.Reduce the proteic activity of PII and mean that reduction promotes the function of adenylylation with ATase.
Except that active situation about being eliminated fully, reduce the proteic activity of PII and mean that this coryneform PII protein-active is lower than coryneform bacteria wild strain or non-modification bacterial strain.For example, in each cell PII protein molecular reduced number bacterial strain, bacterial strain that the PII albumen specific activity of every PII protein molecular has reduced etc. is all included.The contrast usefulness the coryneform example of wild-type comprise, for example, brevibacterium ATCC 13869.Isolated the proteic glnB gene of coding coryneform bacteria PII, and the adenylylation that shows GS can weaken (seeing FEMS MicrobiologyLetters, 303-310, (173) 1999) by the disappearance of gene to the inhibition of GS.
GlnB gene that early the evidence suggests brevibacterium proteic gene of PII of encoding exactly.(seeing EP1229121).The nucleotide sequence of group and coded amino acid thereof are seen SEQ ID NO:23, and aminoacid sequence is seen SEQ ID NO:24.
In the present invention, as the PII albumen that reduces active target, its aminoacid sequence can be included on one or more sites of aforementioned PH Argine Monohydrochloride sequence and lack, substitutes, inserts or add one or more amino-acid residue.
The number change of " one or more " amino-acid residue depends on the position of the amino-acid residue on the protein three-dimensional structure and amino acid whose type.It is the reasons are as follows.That is, some amino acid is the height homology each other, and the difference between them is not enough to proteinic three-dimensional structure is produced remarkable influence.Thereby, the proteic mutant of PII of the present invention can and constitute between the proteic whole amino-acid residues of PII and has 30-50% or higher, preferred 50-70% or higher, more preferably 80-90% or higher, most preferably 95% or higher homology, and have the proteic activity of PII.
Coding and the aforementioned PII albumen DNA of same protein basically comprise that those can have the proteic active DNA of PII with glnB gene recombination and coded albumen under stringent condition." stringent condition " comprises such condition, under this condition, DNA with high homology, for example 50% or higher homology, preferred 70% or higher homology, more preferably 80% or higher homology, most preferably 90% or the DNA of higher homology can hybridize each other, can not hybridize each other but homology is lower than the DNA of above-mentioned level.Perhaps, stringent condition comprises such condition: under this condition, DNA under representative condition corresponding to Southern hybridization, i.e. 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS can be hybridized under 60 ℃ each other.
The example that reduces coryneform bacteria PII protein-active method comprises: for example, with mutagenic compound commonly used in uviolizing or the mutagenic treatment such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid processing coryneform bacteria, pick out then and show as the mutant strain that the PII protein-active reduces.Except sudden change is handled, showing as coryneform bacteria that the PII protein-active reduces can also be available from following method: the partial sequence by disappearance glnB gene makes the PII protein coding gene (glnB) after modifying can't generate the PII albumen of normal function, transform coryneform bacteria with the DNA that contains this glnB gene (absence type glnB gene), thereby make glnB gene on the reorganization destruction karyomit(e) takes place between the glnB gene on above-mentioned absence type glnB gene and the karyomit(e).
These gene disruptions to the glnB gene all can adopt aforementioned gene disruption method to the glnE gene.
(4) modification of nitrogen metabolism mechanism
When nitrogen metabolism is regulated the albumen cisco unity malfunction, also can eliminate the activity control of utilizing the GS adenylylation.
" nitrogen metabolism adjusting albumen " be a kind of with GS become in the inactive state mechanism the factor; this mechanism has adopted the method (adenylylation of glutamine synthetase is the major portion of this nitrogen metabolism regulation mechanism) of the tyrosine residues in the above-mentioned GS aminoacid sequence of adenylylation, and comprises a positive divisor and a negative factor.Positive divisor can increase the activity of GS in the born of the same parents, and the negative factor can reduce the activity of GS in the born of the same parents.Nitrogen metabolism is regulated albumen can not only regulate GS, can also regulate ammonium ion mix gene (amt, amtB).Along with the increase of extracellular ammonium concentration, nitrogen metabolism is regulated albumen and has been reduced the activity of mixing gene such as amt and amtB etc., thereby has suppressed mixing of ammonium ion.
As everyone knows; in intestinal bacteria; when the density loss of glutamine in the born of the same parents, nitrogen metabolism is regulated proteic positive divisor---NRI, and regulates glnD gene (this genes encoding Uridyl transferase (UTase)) expression promoter and combines; to increase the amount that glnD expresses; the proteic increase of uridine acidylate PII promoted the adenylylation that goes of ATase, thereby the activity that has increased GS (is seen Mol.Microbaiology; (1998) (2) 29,431-447).
As everyone knows; in subtilis; when the density loss of glutamine in the born of the same parents; nitrogen metabolism is regulated the proteic negative factor---TnrA or GlnR; dissociate from regulating glnD gene (this genes encoding Uridyl transferase (UTase)) expression promoter, and increase the amount that glnD expresses, the proteic increase of uridine acidylate PII; promote the adenylylation that goes of ATase, thereby increased the activity of GS.
Utilize the nitrogen metabolism controlling mechanism of GS adenylylation, can regulate proteic mode with the modification nitrogen metabolism and modify, thereby increase the activity of GS consistently as main mechanism.When the nitrogen metabolism regulatory gene is a positive divisor, can increase the activity of GS with the active method of enhancement factor consistently, when it is a negative factor, can increase the activity of GS consistently with the method that reduces factor active.
In coryneform bacteria, be that the nitrogen metabolism regulation mechanism of major portion is controlled with the product (AmtR) of amtR gene with the GS adenylylation, this product is a kind of nitrogen metabolism negative regulator (seeing Mol.Microbiol., 37 (4): 964-77, in August, 2000).Thereby, thereby can make the mode of the AmtR effect of can not bringing into normal play increase the activity of GS by modifying the amtR gene.The implication of " AmtR can not bring into normal play effect " is meant, compares with coryneform wild strain or unmodified bacterial strain, and the loss of activity of AmtR or reduction, and caused the active increase of GS.
The amtR nucleotide sequence of brevibacterium and coded amino acid thereof are seen SEQ ID NO:21, and aminoacid sequence is seen SEQ ID NO:22.
In the present invention, as the AmtR that reduces active target, its aminoacid sequence can comprise the disappearance of one or more amino-acid residue on one or more sites of aforementioned AmtR aminoacid sequence, substitute, inserts or add.
The number change of " one or more " amino-acid residue depends on the position of amino-acid residue on protein three-dimensional structure and the type of amino-acid residue.But the mutant of AmtR of the present invention can be to have 30-50% or higher with whole amino-acid residues of forming AmtR, preferred 50-70% or higher, more preferably 80-90% or higher, most preferably 95% or higher homology and have the active protein of AmtR.
The substantially the same proteic DNA with aforementioned AmtR that encodes comprises that those have the active DNA of AmtR with amtR gene recombination and coded albumen under stringent condition.Above-mentioned " stringent condition " is meant under this condition, forms so-called specific hybrid body, and do not form the non-specific hybridization body.For example, stringent condition comprises such condition, under this condition, DNA with high homology, for example 50% or higher homology, preferred 70% or higher homology, more preferably 80% or higher homology, most preferably 90% or the DNA of higher homology can hybridize each other, can not hybridize each other but homology is lower than the DNA of above-mentioned level.Perhaps, stringent condition comprises such condition: under this condition, DNA under representative condition corresponding to Southern hybridization, i.e. 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS can be hybridized under 60 ℃ each other.
Modifying coryneform bacteria comprises can not the bring into normal play example of functional method of its AmtR, for example, with uviolizing or with mutagenic compound commonly used, for example, N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid, handle coryneform bacteria, screening has the active mutant strain of the AmtR of reduction then.Except sudden change is handled, showing as the active coryneform bacteria that reduces of AmtR can also be available from following method: the partial sequence by disappearance amtR gene makes the AmtR encoding gene amtR after modifying can't generate the AmtR of normal function, transform coryneform bacteria with the DNA that contains this amtR gene (absence type amtR gene), thereby make amtR gene on the reorganization destruction karyomit(e) takes place between the amtR gene on above-mentioned absence type amtR gene and the karyomit(e).
These gene disruptions to the AmtR gene all can adopt aforementioned gene disruption method to the glnE gene.
The method that reduces or eliminates the GS adenylylation comprehensively two or more following methods realizes: screening can not be reduced the ATase activity by the GS mutant of adenylylation, reduces the PII protein-active and modifies above-mentioned nitrogen metabolism and regulate albumen.
Utilize microorganisms producing L-arginine provided by the invention or L-Methionin
Cultivate the coryneform bacteria that aforesaid method obtains, gather and collect L-arginine or L-Methionin in the substratum, can produce this two seed amino acid efficiently.
Can adopt ordinary method to cultivate, adopt typical culture, wherein contain carbon source, nitrogenous source, mineral salt and needed such as micro-organic nutrient substances such as amino acid, VITAMIN.In addition, synthetic or natural substratum all can adopt.As long as bacterial strain can utilize to cultivate, any carbon source and nitrogenous source all can adopt.
With regard to carbon source, such as glucose, glycerine, fructose, sucrose, maltose, seminose, semi-lactosi, starch hydrolyzates, carbohydrates such as molasses, and such as organic acids such as acetic acid, citric acids, all can adopt.In addition, alcohols such as ethanol also can be used in combination separately or with other carbon sources.
With regard to organic micro-nutrients, amino acid, VITAMIN, lipid acid, nucleic acid contain materials such as peptone, casamino acids, yeast extract, soybean protein degradation production and all can adopt.When growth needs one seed amino acid of certain auxotrophic mutant or similarly during material, preferably add the nutrition of these needs.
With regard to mineral salt, phosphoric acid salt, magnesium salts, calcium salt, molysite, manganese salt or the like all can adopt.
Culture condition is preferably aerobic.Culture temperature is controlled at 20-45 ℃, and pH 5-9 when pH descends in the culturing process, can add lime carbonate or the alkali such as ammonia is neutralized.After 10-120 hour, will gather a large amount of L-arginine or L-Methionin with the aforesaid method cultivation in the substratum.
After cultivating end, can adopt ordinary method from fermentation culture, to collect L-arginine or L-Methionin.For example, after the removal of the cell in the nutrient solution, again nutrient solution is concentrated until the L-glutaminate crystallization, just can collect L-arginine or L-Methionin.
Embodiment
Below, in conjunction with non-limiting example the present invention is done more specifically explanation.
Reference example 1: the coryneform bacteria that makes up arginine repressor defective
(1) structure is used to destroy the argR plasmid
Chromosomal DNA with brevibacterium flavum wild type strain 2247 bacterial strains (AJ14067) carries out pcr amplification as template, and the primer is for having the oligonucleotide of nucleotide sequence shown in SEQ ID NO:1 (it is corresponding to Nucleotide of SEQ ID NO:15 numbering 4-28) and SEQ ID NO:2 (numbering the sequence complementation of 4230-4211 with the Nucleotide of SEQ ID NO:15).PCR circulation 30 times at every turn 98 ℃ of reactions 10 seconds, 58 ℃ of reactions 1 minute, 72 ℃ of reactions 3 minutes, has been adopted Pyrobest archaeal dna polymerase (Takara Shuzo).Amplified fragments is inserted on the SmaI site of cloning vector pHSG399 multiple clone site.
In order to remove the whole ORF of coding arginine repressor from the dna fragmentation that inserts, it is primer that PCR has adopted the oligonucleotide with nucleotide sequence shown in SEQ ID NO:3 (corresponding to the Nucleotide of SEQ ID NO:15 numbering 2372-2395) and SEQ ID NO:4 (numbering the sequence complementation of 1851-1827 with the Nucleotide of SEQ ID NO:15), is template with the pHSG399 that has inserted amplified fragments.The PCR product oneself connect, thereby obtained pssER.
Next, the fragment that obtains with Restriction enzyme Sma I and SalI digestion pssER and SmaI and the postdigestive temperature sensitive plasmid of SalI pSFKT2 (are seen US6,303,383) connect, obtain a kind of plasmid pssERT that is used to destroy argR, its self-replicating ability in coryneform bacteria becomes responsive to temperature type.
(2) obtain the coryneform bacteria of arginine repressor defective with homologous recombination method
The pssERT plasmid of above-mentioned acquisition is imported in the wild type strain 2256 (ATCC13869) of brevibacterium.Plasmid electricity consumption impulse method imports (seeing Japanese patent laid-open No.2-207791).Because this plasmid has temperature sensitive self-replicating ability in brevibacterium, so have only the bacterial strain that this plasmid mixes in the karyomit(e) could be screened be come out with homologous recombination method, its as the bacterial strain of anti-kantlex in 34 ℃ of screened come out, plasmid reproducible not in this temperature.As the bacterial strain of anti-kantlex, with containing 25 μ g/ml kanamycin C M2G plate (the poly-peptone that contains 10g/L, the yeast extracts of 10g/L, the glucose of 5g/L, the sodium-chlor of 5g/L, the agar of 15g/L are in 1L water, and pH is 7.2) filter out and mixed the bacterial strain that destroys the argR plasmid on the karyomit(e).In this stage, derive from the both sides that chromosomal natural argR gene and the argG gene that derives from plasmid (this plasmid removing ORF) are series at karyomit(e) plasmid part.
Then, allow above-mentioned recombinant bacterial strain homologous recombination once more, filter out kantlex responsive type bacterial strain at 34 ℃, plasmid reproducible not under this temperature, thus obtained lacking the bacterial strain of certain argR gene.Comprise the bacterial strain that has kept natural argR gene on the karyomit(e) in these bacterial strains, also comprised the bacterial strain that has kept the argR gene after destroying on the karyomit(e).From these bacterial strains, filter out the bacterial strain that only has the argR gene after the destruction again.Whether ruined method is as follows to determine argR gene in the karyomit(e): in the karyomit(e)s of 34 ℃ of preparation kantlex responsive type bacterial strains, with this karyomit(e) is that template is carried out PCR, with the oligonucleotide sequence with nucleotide sequence shown in SEQ ID NO:1 and 2 is primer, and whether prove conclusively this PCR product then is that template is carried out the short about 600bp of product that similar pcr amplification obtains than using the karyomit(e) from parent strain.
The PCR product that destroys bacterial strain with the argR of above-mentioned selection directly checks order, and can confirm that whether the argR gene is destroyed on demand, so just obtained the 2256DR bacterial strain.This bacterial strain is compared with its parent, and the arginic output of its L-has had significant raising (seeing US2002045223 A1).
Embodiment 1: make up adenylyl transferase (GlnE) defective bacterial strain
(1) preparation is in order to remove the used plasmid of GlnE
The glnE sequence of brevibacterium flavum ATCC 14067 clear already (seeing EP1229121 A2).Nucleotides sequence with report is classified the basis as, has synthesized the primer shown in the SEQ ID NOS:5 and 6, with the method for PCR, with the chromosomal DNA of the brevibacterium ATCC 13869 bacterial strains internal sequence of glnE that has been template amplification.
The chromosomal DNA that has prepared brevibacterium ATCC 13869 bacterial strains with bacterial genomes DNA purification kit (Advanced Genetic Technologies Corp.).PCR circulation 30 times at every turn 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 15 seconds, was extended 2 minutes at 72 ℃, and has been adopted PyrobestDNA polysaccharase (Takara Shuzo).
Use the ordinary method purified pcr product, with Blunting test kit (Takara Shuzo) flush endization.(Takara Shuzo) is connected to flush end PCR product on the HincII site of pHSG299 with the Ligation test kit, and is used for the competent cell of transformed into escherichia coli JM109 (Takara Shuzo).The gained cell is plated on and contains 10 μ g/ml IPTG, overnight incubation on the L-substratum of 40 μ g/ml X-Gal and 25 μ g/ml kantlex.Choose white colony, and it is separated into single bacterium colony, finally obtain transformant.
From above-mentioned transformant, prepare plasmid with alkaline process, and prove conclusively the structure of this plasmid.The segmental plasmid called after of glnE part p Δ ATase-299 will have been inserted in the carrier.
But aforesaid p Δ ATase-299 do not comprise any in coryneform bacteria the sequence of self-replicating.Thereby if transform coryneform bacteria with this plasmid, then to mix chromosomal strains expressed because of homologous recombination be transformant to this plasmid, though the frequency that its occurs is extremely low.
(2) p Δ ATase-299 is imported arginine repressor defective bacterial strain, and measure amino acid output
Transform the arginine repressor defective bacterial strain (2256 Δ argR bacterial strain) that reference example 1 obtains by electricimpulse method (seeing Japanese patent laid-open No.2-207791) with plasmid p Δ ATase-299.With the transformant that the obtains cultivation of going down to posterity, and use the kalamycin resistance acquisition transformant that serves as a mark, thereby obtain the kantlex sensitive strain.Near in the bacterial strain glnE gene sequence pcr amplification, and destroy its glnE gene, with the ruined bacterial strain of glnE (2256 Δ argR Δ glnE) of guaranteeing to obtain 2256 Δ argR.The process of producing L-arginine and L-Methionin with the transformant 2256 Δ arR Δ glnE that obtained is as described below.
In CM2G plate substratum, cultivate, contain 25 μ g/ml kantlex in the substratum, be inoculated into then and contain 40g glucose, 65g (NH
4)
2SO
4, 1g KH
2PO
4, 0.4g MgSO
4.7H
2O, 0.01gFeSO
4, 0.01g MnSO
4, 50 μ g VB1-HCl, 50 μ g vitamin Hs, 45mg (quantity of N) soybean hydrolyzate and 50gCaCO
3In the substratum of in the 1L pure water, (regulating pH to 7.0) with KOH, shaking culture in 31.5 ℃, the sugar part in exhausting substratum, thus obtain 2256 Δ argR Δ glnE cells.
After cultivating end, suitably dilute nutrient solution, the amount of the L-arginine (Arg) that wherein gathers with liquid-phase chromatographic analysis, analyze the amount of the L-Methionin (Lys) wherein gather and the L-L-glutamic acid (Glu) that gathers with Biotech analyser (Asahi Chemical Industry), the L-glutaminate (Gln) that wherein gathers with liquid-phase chromatographic analysis and the amount of accumulation N-acetylglutamat.Analytical results sees Table 1.
Also analyzed the GS activity of above-mentioned each bacterial strain.GS activation analysis method is as follows, is containing 100mM imidazoles-HCL (pH7.0), 0.1mM NH
4CL, 1mM MnCl
2, 1mM phosphoenolpyruvic acid, 0.3mM NADH, the 10U serum lactic dehydrogenase, the 25U pyruvate kinase, 1mM ATP adds thick enzyme solution in the solution of 10mM MSG, in 30 ℃ of variations of measuring 340nm place light absorption ratio, this method sees Journalof Fermentation and Bioengineering, Vol.70, No.3 for details, 182-184,1990.When measuring blank the group, use the above-mentioned reaction soln that does not contain MSG.The preparation method of thick enzyme solution is as follows, and earlier with above-mentioned medium centrifugal isolated cell, the cell that obtains is used the ultrasonic disruption cell again with 100mM imidazoles-HCL (pH7.0) washing, removes not broken cell with centrifuging, obtains thick enzyme solution thereby separate.Measure proteinic concentration in the thick enzyme solution with Protein Assay (Bio-Rad), wherein adopt bovine serum albumin as standard model.The result is as shown in table 2.
Table 1
| Strains A rg Lys Glu Gln N-acetylglutamat (g/L) is (g/L) (g/L) (g/L) (g/L) |
| 2256ΔargR?????????2.28???0.81???0.32???0.19????0.0153 2256ΔargRΔglnE???3.18???1.32???0.4????0.2?????0.0381 |
Table 2
| Bacterial strain GS activity (nmol/min/mg) |
| 2256ΔargR???????????51.2 2256ΔargRΔglnE?????123.1 |
(2256 Δ argR) compares with parent strain, and the L-arginine of GlnE defective bacterial strain and L-Methionin gather and obtained raising.Compare with parent strain, the GSization activity of GlnE defective bacterial strain has improved 2.4 times.Also observe in addition, also be improved as the N-acetylglutamat of L-arginine precursor and the output of L-glutamic acid.
The private numbering of 2256 Δ argR Δ glnE bacterial strains is AJ110145, be preserved in Industrial Technology Institute life engineering Industrial Technology Research Institute of Ministry of International Trade and Industry and (now be independent administration mechanism, Independent Administrative Leged Industrial Technology Complex Inst, biological (the Tsukuba Central 6 of depositary institution of international monopoly, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postcode: 305-5466), preservation date is on February 18th, 2003, and preserving number is FERM P-19216.Then, transfer international preservation according to budapest treaty on February 19th, 2004, preserving number is FERM BP-08630.
The assessment of the bacterial strain that embodiment 2:GS adenylylation site is modified
(1) makes up the plasmid that GlnA adenylylation site is modified
The adenylylation site of coryneform bacteria glnA gene product (GlnA) clear already (seeing FEMSMicrobiology Letters, 303-310, (173) 1999).With the glnA gene on the glnA gene substituted dyeing body in modified GlnA adenylylation site, just obtained the bacterial strain that GlnA adenylylation site is modified.To be described its detailed process below.
At first, with the method for PCR, be template with the chromosomal DNA of brevibacterium ATCC 13869 bacterial strains, be primer with synthetic DNA shown in SEQ ID NOS:7 and 8, obtained the N-end side amplified production of glnA gene.In addition, in order to obtain the C-end side amplified production of glnA gene, PCR is a template with the chromosomal DNA of brevibacterium ATCC 13869 bacterial strains, is primer with synthetic DNA shown in SEQ ID NOS:9 and 10.Because imported mispairing in the sequence shown in SEQ ID NOS:8 and 9, so the terminal portions of each amplified production has all imported sudden change.Specifically, this sudden change makes the tyrosine residues on 405 be replaced by the L-phenylalanine.
In order to have obtained to have imported the glnA gene fragment of sudden change, with the aforementioned gene product of the N-of the glnA of balanced mix and C-end side as template, with SEQ ID NOS; Synthetic DNA shown in 10 and 11 is a primer, obtains the glnA gene amplification product that sudden change has been introduced in the adenylylation site through PCR.PCR product ordinary method purifying is with HincII digestion, the HincII site of inserting pHSG299 (Takar Shuzo) then.This plasmid is named as pGSA2.
Because above-mentioned pGSA2 do not contain make its can be in coryneform bacteria the zone of self-replicating, so when transforming coryneform bacteria with this plasmid, it is transformant that this plasmid mixes chromosomal strains expressed because of homologous recombination, though the frequency that its occurs is extremely low.
(2) pGSA2 is imported arginine repressor defective bacterial strain, and measure amino acid output
(see Japanese Patent with the electricimpulse method, Te Kaiping No.2-207791) the plasmid pGSA2 with high density transforms, the ArgR defective bacterial strain 2256 Δ R that come from brevibacterium ATCC 13869 that reference example 1 is described are that mark has obtained transformant with anti-kantlex.Then, with the transformant that the obtains cultivation of going down to posterity, to obtain the kantlex sensitive strain.Measure the glnE gene order of each transformant, with adenylylation site in the sequence by from transformant called after 2256 Δ argRAde that should the zone replaced among the glnA of pGSA2.Obtain L-arginine and L-Methionin with 2256 Δ argR bacterial strains and 2256 Δ argRAde strain culturing, with embodiment 1, the method for (2) is measured the GS enzymic activity.The result is shown in table 3,4.
Table 3
| Strains A rg Lys Glu Gln N-acetylglutamat (g/L) is (g/L) (g/L) (g/L) (g/L) |
| 2256ΔargR??????2.28????0.81????0.32????0.19????0.0153 2256ΔargRAde???2.88????1.48????0.49????0.31????0.314 |
Table 4
| Bacterial strain GS activity (nmol/min/mg) |
| ?2256ΔargR?????????51.2 ?2256ΔargRAde??????135.1 |
Compare with 2256 Δ argR, the L-arginine and the L-lysine production of 2256 Δ argRAde bacterial strains have obtained raising.Also observe in addition, also be improved as the output of L-L-glutamic acid, L-glutaminate and the N-acetylglutamat of L-arginine precursor.The specific activity of GS has improved about 2.6 times.
The private numbering of 2256 Δ argRAde bacterial strains is AJ110146, be preserved in Industrial Technology Institute life engineering Industrial Technology Research Institute of Ministry of International Trade and Industry and (now be independent administration mechanism, Independent Administrative Leged Industrial Technology Complex Inst, biological (the Tsukuba Central 6 of depositary institution of international monopoly, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postcode: 305-5466), preservation date is on February 18th, 2003, and preserving number is FERM P-19217.Then, transfer international preservation according to budapest treaty on February 19th, 2004, preserving number is FERM BP-08631.
Embodiment 3: obtain and estimate the AmtR deficient strain
(1) preparation is used to remove the plasmid of AmtR
Be used to remove the plasmid of corynebacteria A mtR gene product (AmtR), be prepared as follows.
At first, extract the chromosomal DNA of brevibacterium ATCC 13869, and it is carried out PCR as template with the synthetic DNA shown in SEQ ID NOS:13 and 14.With the dna fragmentation flush endization that obtains, and be inserted into the HincII site of pHSG299 (Taka Shuzo).Resulting plasmid is named as p Δ AmtRT.
Above-mentioned p Δ AmtR do not contain make its can be in the coryneform bacteria cell sequence of self-replicating, so after coryneform bacteria was transformed by this plasmid, it was transformant that this plasmid mixes chromosomal strains expressed because of homologous recombination, though the frequency that its occurs is extremely low.
(2) p Δ AmtR is imported arginine repressor defective bacterial strain, and measure the cultivation of bacterial strain
Method with reference example 1 obtains arginine repressor defective bacterial strain (2256 Δ argR bacterial strain), and this bacterial strain transforms with p Δ AmtR, is that mark has obtained transformant with anti-kantlex.
Then, the conversion strain passage that obtains is cultivated, to obtain the kantlex sensitive strain.Near each transformant AmtR gene sequence is checked order, and confirm to have destroyed the amtR gene, thereby the amtR that has obtained 2256 Δ argR destroys bacterial strain (2256 Δ argR Δ AmtR).Obtain L-arginine and L-Methionin with the transformant 2256 Δ argR Δ amtR strain culturing that obtain, with embodiment 1, the method for (2) is measured its output.The result is shown in table 5,6.
Table 5
| Strains A rg Lys Glu Gln N-acetylglutamat (g/L) is (g/L) (g/L) (g/L) (g/L) |
| 2256ΔargR????????2.28????0.81????0.32????0.19????0.0153 2256ΔargRΔAmtR??3.16????1.48????0.45????0.41????0.0296 |
Table 6
| Bacterial strain GS activity (nmol/min/mg) |
| ??2256ΔargR???????????51.2 ??2256ΔargRΔAmtR?????132.3 |
Compare with 2256 Δ argR, L-arginine and the L-lysine production of 2256 Δ argR Δ amtR have obtained raising.Also observe in addition, also be improved as the output of L-glutamic acid, N-acetylglutamat and the glutamine of L-arginine precursor.The activity specific of GS has improved about 2.6 times.
The private numbering of 2256 Δ argR Δ AmtR bacterial strains is AJ110144, is preserved in Industrial Technology Institute life engineering Industrial Technology Research Institute of Ministry of International Trade and Industry and (now is independent administration mechanism, Independent Administrative Leged Industrial Technology Complex Inst, the biological depositary institution of international monopoly (Tsukuba Central 6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postcode: 305-5466), preservation date is 2003, February 18, preserving number is FERM P-19215.Then, transfer international preservation according to budapest treaty on February 19th, 2004, preserving number is FERM BP-08629.
Although the present invention is described in detail with embodiment preferred, those skilled in the art can make various changes obviously, adopts equivalent, and does not depart from the scope of the present invention.Aforesaid each piece document comprises the foreign priority file, and JP 2003-56129 is incorporated herein by reference in this integral body.
[sequence table explanation]
The primer of SEQ ID NO:1argR gene amplification
The primer of SEQ ID NO:2argR gene amplification
SEQ ID NO:3 be used for increasing contain the argR gene plasmid except that argR gene ORF the part primer
SEQ ID NO:4 be used for increasing contain the argR gene plasmid except that argR gene ORF the part primer
SEQ ID NO:5 is used to destroy the primer N of glnR
SEQ ID NO:6 is used to destroy the primer C of glnR
SEQ ID NO:7glnA adenylylation the one PCR primer NN
SEQ ID NO:8glnA adenylylation the one PCR primer NC
SEQ ID NO:9glnA adenylylation the one PCR primer CN
SEQ ID NO:10glnA adenylylation the one PCR primer CC
SEQ ID NO:11glnA the 2nd PCR primer N
SEQ ID NO:12glnA the 2nd PCR primer C
SEQ ID NO:13 is used to destroy the primer N of amtR
SEQ ID NO:14 is used to destroy the primer C of amtR
The nucleotide sequence of SEQ ID NO:15argR gene
The coded aminoacid sequence of dna fragmentation above the SEQ ID NO:16
The nucleotide sequence of SEQ ID NO:17glnE gene
The coded aminoacid sequence of dna fragmentation above the SEQ ID NO:18
The nucleotide sequence of SEQ ID NO:19glnA gene
The coded aminoacid sequence of dna fragmentation above the SEQ ID NO:20
The nucleotide sequence of SEQ ID NO:21amtR gene
The coded aminoacid sequence of dna fragmentation above the SEQ ID NO:22
The nucleotide sequence of SEQ ID NO:23glnB gene
The coded aminoacid sequence of dna fragmentation above the SEQ ID NO:24
Sequence table
<110>??Ajinomoto，Co.，Inc.
＜120〉method by fermentative production L-arginine or L-Methionin
<130>
<150>??JP?2003-056129
<151>??2003-03-03
<160>??24
<170>??PatentIn?Ver.2.0
<210>??1
<211>??25
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the primer that is used for PCR
<400>??1
cccgggtttt?cttctgcaac?tcggg????????????????????????????????????????25
<210>??2
<211>??25
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the primer that is used for PCR
<400>??2
gtcgacaagc?tcggttgttc?ccagc????????????????????????????????????????25
<210>??3
<211>??24
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the primer that is used for PCR
<400>??3
cccctagttc?aaggcttgtt?aatc?????????????????????????????????????????24
<210>??4
<211>??25
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the primer that is used for PCR
<400>??4
gtcttacctc?ggctggttgg?ccagc????????????????????????????????????????25
<210>??5
<211>??23
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence; Primer
<400>??5
agaactacga?gtccgccttt?ttg??????????????????????????????????????????23
<210>??6
<211>??21
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??6
cgatcaccag?caacccacgc?a????????????????????????????????????????????21
<210>??7
<211>??22
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??7
cttcccagta?gcaccatacg?ac???????????????????????????????????????????22
<210>??8
<211>??26
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??8
ctggtggcag?ttcgaagagg?tccttg???????????????????????????????????????26
<210>??9
<211>??26
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??9
ggacaaggac?ctcttcgaac?tgccag???????????????????????????????????????26
<210>??10
<211>??26
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??10
cggcgagacc?gtcgattggg?aggagc???????????????????????????????????????26
<210>??11
<211>??22
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??11
gtagcacctt?acgaccaaac?cg???????????????????????????????????????????22
<210>??12
<211>??20
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??12
ggagccggtc?gacgaggagc??????????????????????????????????????????????20
<210>??13
<211>??23
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??13
gccccgggca?ggcaagaatc?ctc??????????????????????????????????????????23
<210>??14
<211>??21
<212>??DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer
<400>??14
tccccgggag?gctctctgcg?g????????????????????????????????????????????21
<210>??15
<211>??4235
<212>??DNA
＜213〉brevibacterium flavum (Brevibacterium flavum)
<220>
<221>??CDS
<222>??(1852)..(2364)
<400>??15
aaacccgggt?tttcttctgc?aactcgggcg?ccgaagcaaa?cgaggctgct?ttcaagattg??60
cacgcttgac?tggtcgttcc?cggattctgg?ctgcagttca?tggtttccac?ggccgcacca??120
tgggttccct?cgcgctgact?ggccagccag?acaagcgtga?agcgttcctg?ccaatgccaa??180
gcggtgtgga?gttctaccct?tacggcgaca?ccgattactt?gcgcaaaatg?gtagaaacca??240
acccaacgga?tgtggctgct?atcttcctcg?agccaatcca?gggtgaaacg?ggcgttgttc??300
cagcacctga?aggattcctc?aaggcagtgc?gcgagctgtg?cgatgagtac?ggcatcttga??360
tgatcaccga?tgaagtccag?actggcgttg?gccgtaccgg?cgatttcttt?gcacatcagc??420
acgatggcgt?tgttcccgat?gtggtgacca?tggccaaggg?acttggcggc?ggtcttccca??480
tcggtgcttg?tttggccact?ggccgtgcag?ctgaattgat?gaccccaggc?aagcacggca??540
ccactttcgg?tggcaaccca?gttgcttgtg?cagctgccaa?ggcagtgctg?tctgttgtcg??600
atgacgcttt?ctgcgcagaa?gttacccgca?agggcgagct?gttcaaggta?cttcttgcca??660
aggttgacgg?cgttgtagac?gtccgtggca?ggggcttgat?gttgggcgtg?gtgctggagc??720
gcgacgtcgc?aaagcaagct?gttcttgatg?gttttaagca?cggcgttatt?ttgaatgcac??780
cggcggacaa?cattatccgt?ttgaccccgc?cgctggtgat?caccgacgaa?gaaatcgcag??840
acgcagtcaa?ggctattgcc?gagacaatcg?cataaaggac?ttaaacttat?gacttcacaa??900
ccacaggttc?gccatttcct?ggctgatgat?gatctcaccc?ctgcagagca?ggcagaggtt??960
ttgaccctag?ccgcaaagct?caaggcagcg?ccgttttcgg?agcgtccact?cgagggacca??1020
aagtccgttg?cagttctttt?tgataagact?tcaactcgta?ctcgcttctc?cttcgacgcg??1080
ggcatcgctc?atttgggtgg?acatgccatc?gtcgtggatt?ccggcagctc?acagatgggt??1140
aagggcgaga?ccctgcagga?caccgcagct?gtattgtccc?gctacgtgga?agcaattgtg??1200
tggcgcacct?acgcacacag?caatttccac?gccatggcgg?agacgtccac?tgtgccgctg??1260
gtgaactcct?tgtccgatga?tctgcaccca?tgccagattc?tggctgatct?gcagaccatc??1320
gtggaaaacc?tcagccctga?agaaggccca?gcaggcctta?agggtaagaa?ggctgtgtac??1380
ctgggcgatg?gcgacaacaa?catggccaac?tcctacatga?ttggctttgc?caccgcgggc??1440
atggatattt?ccatcatcgc?tcctgaaggg?ttccagcctc?gtgcggaatt?cgtggagcgc??1500
gcggaaaagc?gtggccagga?aaccggcgcg?aaggttgttg?tcaccgacag?cctcgacgag??1560
gttgccggcg?ccgatgttgt?catcaccgat?acctgggtat?ccatgggtat?ggaaaacgac??1620
ggcatcgatc?gcaccacacc?tttcgttcct?taccaggtca?acgatgaggt?catggcgaaa??1680
gctaacgacg?gcgccatctt?cctgcactgc?cttcctgcct?accgcggcaa?agaagtggca??1740
gcctccgtga?ttgatggacc?agcgtccaaa?gttttcgatg?aagcagaaaa?ccgcctccac??1800
gctcagaaag?cactgctggt?gtggctgctg?gccaaccagc?cgaggtaaga?c?atg?tct???1857
Met?Ser
1
ctt?ggc?tca?acc?ccg?tca?aca?ccg?gaa?aac?tta?aat?ccc?gtg?act?cgc????1905
Leu?Gly?Ser?Thr?Pro?Ser?Thr?Pro?Glu?Asn?Leu?Asn?Pro?Val?Thr?Arg
5??????????????????10??????????????????15
act?gca?cgc?caa?gct?ctc?att?ttg?cag?att?ttg?gac?aaa?caa?aaa?gtc????1953
Thr?Ala?Arg?Gln?Ala?Leu?Ile?Leu?Gln?Ile?Leu?Asp?Lys?Gln?Lys?Val
20??????????????????25??????????????????30
acc?agc?cag?gta?caa?ctg?tct?gaa?ttg?ctg?ctg?gat?gaa?ggc?atc?gat????2001
Thr?Ser?Gln?Val?Gln?Leu?Ser?Glu?Leu?Leu?Leu?Asp?Glu?Gly?Ile?Asp
35??????????????????40??????????????????45??????????????????50
atc?acc?cag?gcc?acc?ttg?tcc?cgg?gat?ctc?gat?gaa?ctc?ggt?gca?cgc????2049
Ile?Thr?Gln?Ala?Thr?Leu?Ser?Arg?Asp?Leu?Asp?Glu?Leu?Gly?Ala?Arg
55??????????????????60??????????????????65
aag?gtt?cgc?ccc?gat?ggg?gga?cgc?gcc?tac?tac?gcg?gtc?ggc?cca?gta????2097
Lys?Val?Arg?Pro?Asp?Gly?Gly?Arg?Ala?Tyr?Tyr?Ala?Val?Gly?Pro?Val
70??????????????????75??????????????????80
gat?agc?atc?gcc?cgc?gaa?gat?ctc?cgg?ggt?ccg?tcg?gag?aag?ctg?cgc????2145
Asp?Ser?Ile?Ala?Arg?Glu?Asp?Leu?Arg?Gly?Pro?Ser?Glu?Lys?Leu?Arg
85??????????????????90??????????????????95
cgc?atg?ctt?gat?gaa?ctg?ctg?gtt?tct?aca?gat?cat?tcc?ggc?aac?atc????2193
Arg?Met?Leu?Asp?Glu?Leu?Leu?Val?Ser?Thr?Asp?His?Ser?Gly?Asn?Ile
100?????????????????105?????????????????110
gcg?atg?ctg?cgc?acc?ccg?ccg?gga?gct?gcc?cag?tac?ctg?gca?agt?ttc????2241
Ala?Met?Leu?Arg?Thr?Pro?Pro?Gly?Ala?Ala?Gln?Tyr?Leu?Ala?Ser?Phe
115?????????????????120?????????????????125?????????????????130
atc?gat?agg?gtg?ggg?ctg?aaa?gaa?gtc?gtt?ggc?acc?atc?gct?ggc?gat????2289
Ile?Asp?Arg?Val?Gly?Leu?Lys?Glu?Val?Val?Gly?Thr?Ile?Ala?Gly?Asp
135?????????????????140?????????????????145
gac?acc?gtt?ttt?gtt?ctc?gcc?cgt?gat?ccg?ctc?aca?ggt?aaa?gaa?cta????2337
Asp?Thr?Val?Phe?Val?Leu?Ala?Arg?Asp?Pro?Leu?Thr?Gly?Lys?Glu?Leu
150?????????????????155?????????????????160
ggt?gaa?tta?ctc?agc?ggg?cgc?acc?act?taaagcgccc?ctagttcaag??????????2384
Gly?Glu?Leu?Leu?Ser?Gly?Arg?Thr?Thr
165?????????????????170
gcttgttaat?cgcttgttaa?tgcaggcagg?taaggtataa?cccgagtgtt?ttttcgagga??2444
ataccaaccc?tttcaacaca?ataattttct?ttaaacatcc?ttgctgtcca?ccacggctgg??2504
caaggaactt?aaaatgaagg?agcacacctc?atgactaacc?gcatcgttct?tgcatactcc??2564
ggcggtctgg?acaccactgt?ggcaattcca?tacctgaaga?agatgattga?tggtgaagtc??2624
atcgcagttt?ctctcgacct?gggccagggt?ggagagaaca?tggacaacgt?tcgccagcgt??2684
gcattggatg?ccggtgcagc?tgagtccatc?gttgttgatg?caaaggatga?gttcgctgag??2744
gagtactgcc?tgccaaccat?caaggcaaac?ggcatgtaca?tgaagcagta?cccactggtt??2804
tctgcaatct?cccgcccact?gatcgtcaag?cacctcgttg?aggctggcaa?gcagttcaac??2864
ggtacccacg?ttgcacacgg?ctgcactggt?aagggcaacg?accaggttcg?tttcgaggtc??2924
ggcttcatgg?acaccgatcc?aaacctggag?atcattgcac?ctgctcgtga?cttcgcatgg??2984
acccgcgaca?aggctatcgc?cttcgccgag?gagaacaacg?ttccaatcga?gcagtccgtg??3044
aagtccccat?tctccatcga?ccagaacgtc?tggggccgcg?ctattgagac?cggttacctg??3104
gaagatctgt?ggaatgctcc?aaccaaggac?atctacgcat?acaccgagga?tccagctctg??3164
ggtaacgctc?cagatgaggt?catcatctcc?ttcgagggtg?gcaagccagt?ctccatcgat??3224
ggccgtccag?tctccgtact?gcaggctatt?gaagagctga?accgtcgtgc?aggcgcacag??3284
ggcgttggcc?gccttgacat?ggttgaggac?cgtctcgtgg?gcatcaagtc?ccgcgaaatc??3344
tacgaagcac?caggcgcaat?cgcactgatt?aaggctcacg?aggctttgga?agatgtcacc??3404
atcgagcgcg?aactggctcg?ctacaagcgt?ggcgttgacg?cacgttgggc?tgaggaagta??3464
tacgacggcc?tgtggttcgg?acctctgaag?cgctccctgg?acgcgttcat?tgattccacc??3524
caggagcacg?tcaccggcga?tatccgcatg?gttctgcacg?caggttccat?caccatcaat??3584
ggtcgtcgtt?ccagccactc?cctgtacgac?ttcaacctgg?ctacctacga?caccggcgac??3644
accttcgacc?agaccctggc?taagggcttt?gtccagctgc?acggtctgtc?ctccaagatc??3704
gctaacaagc?gcgatcgcga?agctggcaac?aactaagcca?ccttttcaag?catccagact??3764
agaacttcaa?gtatttagaa?agtagaagaa?caccacatgg?aacagcacgg?aaccaatgaa??3824
ggtgcgctgt?ggggcggccg?cttctccggt?ggaccctccg?aggccatgtt?cgccttgagt??3884
gtctccactc?atttcgactg?ggttttggcc?ccttatgatg?tgttggcctc?caaggcacac??3944
gccaaggttt?tgcaccaagc?agagctactt?tctgatgaag?atctagccac?catgctggct??4004
ggtcttgatc?agctgggcaa?ggatgtcgcc?gacggaacct?tcggtccgct?gccttctgat??4064
gaggatgtgc?acggcgcgat?ggaacgcggt?ctgattgacc?gcgttggtcc?tgaggtgggc??4124
ggccgtctgc?gcgctggtcg?ttcccgcaac?gaccaggtgg?caaccctgtt?ccgcatgtgg??4184
gtccgcgacg?cagtgcgcga?catcgcgctg?ggaacaaccg?agcttgtcga?c???????????4235
<210>??16
<211>??171
<212>??PRT
＜213〉brevibacterium flavum
<400>??16
Met?Ser?Leu?Gly?Ser?Thr?Pro?Ser?Thr?Pro?Glu?Asn?Leu?Asn?Pro?Val
1???????????????5??????????????????10??????????????????15
Thr?Arg?Thr?Ala?Arg?Gln?Ala?Leu?Ile?Leu?Gln?Ile?Leu?Asp?Lys?Gln
20??????????????????25??????????????????30
Lys?Val?Thr?Ser?Gln?Val?Gln?Leu?Ser?Glu?Leu?Leu?Leu?Asp?Glu?Gly
35??????????????????40??????????????????45
Ile?Asp?Ile?Thr?Gln?Ala?Thr?Leu?Ser?Arg?Asp?Leu?Asp?Glu?Leu?Gly
50???????????????????55??????????????????60
Ala?Arg?Lys?Val?Arg?Pro?Asp?Gly?Gly?Arg?Ala?Tyr?Tyr?Ala?Val?Gly
65??????????????????70??????????????????75??????????????????80
Pro?Val?Asp?Ser?Ile?Ala?Arg?Glu?Asp?Leu?Arg?Gly?Pro?Ser?Glu?Lys
85??????????????????90??????????????????95
Leu?Arg?Arg?Met?Leu?Asp?Glu?Leu?Leu?Val?Ser?Thr?Asp?His?Ser?Gly
100?????????????????105?????????????????110
Asn?Ile?Ala?Met?Leu?Arg?Thr?Pro?Pro?Gly?Ala?Ala?Gln?Tyr?Leu?Ala
115?????????????????120?????????????????125
Ser?Phe?Ile?Asp?Arg?Val?Gly?Leu?Lys?Glu?Val?Val?Gly?Thr?Ile?Ala
130?????????????????135?????????????????140
Gly?Asp?Asp?Thr?Val?Phe?Val?Leu?Ala?Arg?Asp?Pro?Leu?Thr?Gly?Lys
145?????????????????150?????????????????155?????????????????160
Glu?Leu?Gly?Glu?Leu?Leu?Ser?Gly?Arg?Thr?Thr
165?????????????????170
<210>??17
<211>??3138
<212>??DNA
＜213〉brevibacterium (Brevibacterium lactofermentum)
<220>
<221>??CDS
<222>??(1)..(3138)
<400>??17
atg?tca?gga?ccg?tta?aga?agt?gaa?cgt?aaa?gtc?gtt?ggc?ttt?gtc?aga????48
Met?Ser?Gly?Pro?Leu?Arg?Ser?Glu?Arg?Lys?Val?Val?Gly?Phe?Val?Arg
1?????????????????5??????????????????10??????????????????15
gac?cca?ctg?cca?aaa?gtt?ggt?tct?tta?tcg?ctg?aaa?tct?gag?cat?gcc????96
Asp?Pro?Leu?Pro?Lys?Val?Gly?Ser?Leu?Ser?Leu?Lys?Ser?Glu?His?Ala
20??????????????????25??????????????????30
caa?gca?gat?cta?gag?cat?ttg?ggt?tgg?cgc?aat?gtt?gag?tct?ttg?gat????144
Gln?Ala?Asp?Leu?Glu?His?Leu?Gly?Trp?Arg?Asn?Val?Glu?Ser?Leu?Asp
35??????????????????40?????????????????45
ttg?ttg?tgg?ggc?ttg?tca?ggt?gca?ggc?gat?ccc?gat?gtc?gcg?ctg?aac????192
Leu?Leu?Trp?Gly?Leu?Ser?Gly?Ala?Gly?Asp?Pro?Asp?Val?Ala?Leu?Asn
50??????????????????55??????????????????60
ctt?ctt?att?cgg?ctg?tat?cag?gca?ctt?gaa?gca?atc?ggc?gag?gat?gct????240
Leu?Leu?Ile?Arg?Leu?Tyr?Gln?Ala?Leu?Glu?Ala?Ile?Gly?Glu?Asp?Ala
65??????????????????70??????????????????75??????????????????80
cga?aac?gag?ctt?gat?caa?gag?att?cgc?cag?gat?gaa?gaa?cta?cga?gtc????288
Arg?Asn?Glu?Leu?Asp?Gln?Glu?Ile?Arg?Gln?Asp?Glu?Glu?Leu?Arg?Val
85??????????????????90??????????????????95
cgc?ctt?ttt?gca?ttg?ttg?ggt?ggt?tcc?tcg?gct?gtc?ggt?gat?cac?ttg????336
Arg?Leu?Phe?Ala?Leu?Leu?Gly?Gly?Ser?Ser?Ala?Val?Gly?Asp?His?Leu
100?????????????????105?????????????????110
gtc?gcc?aat?cct?ttg?cag?tgg?aaa?ctc?tta?aaa?ctt?gat?gcg?cca?tcg????384
Val?Ala?Asn?Pro?Leu?Gln?Trp?Lys?Leu?Leu?Lys?Leu?Asp?Ala?Pro?Ser
115?????????????????120?????????????????125
agg?gaa?gag?atg?ttt?cag?gcg?ctg?ctg?gaa?tct?gtg?aaa?gct?cag?cct????432
Arg?Glu?Glu?Met?Phe?Gln?Ala?Leu?Leu?Glu?Ser?Val?Lys?Ala?Gln?Pro
130?????????????????135?????????????????140
gct?gtg?ctt?gag?gtt?gag?gat?ttc?agc?gat?gca?cac?aac?att?gcc?cga????480
Ala?Val?Leu?Glu?Val?Glu?Asp?Phe?Ser?Asp?Ala?His?Asn?Ile?Ala?Arg
145?????????????????150?????????????????155?????????????????160
gac?gat?ttg?agc?acg?cct?ggt?ttt?tac?acg?gct?agt?gtt?acc?ggg?cct????528
Asp?Asp?Leu?Ser?Thr?Pro?Gly?Phe?Tyr?Thr?Ala?Ser?Val?Thr?Gly?Pro
165?????????????????170?????????????????175
gaa?gca?gag?cga?gtc?ttg?aaa?tgg?act?tat?cgc?acg?ttg?ctg?acc?cgg????576
Glu?Ala?Glu?Arg?Val?Leu?Lys?Trp?Thr?Tyr?Arg?Thr?Leu?Leu?Thr?Arg
180?????????????????185?????????????????190
att?gct?gcg?cat?gat?tta?gcg?ggt?acc?tat?ccc?acc?gac?atg?cgg?aga????624
Ile?Ala?Ala?His?Asp?Leu?Ala?Gly?Thr?Tyr?Pro?Thr?Asp?Met?Arg?Arg
195?????????????????200?????????????????205
aaa?ggt?ggc?gat?cct?gtt?ccg?ttt?agc?aca?gtg?acc?atg?cag?ctc?agc????672
Lys?Gly?Gly?Asp?Pro?Val?Pro?Phe?Ser?Thr?Val?Thr?Met?Gln?Leu?Ser
210?????????????????215?????????????????220
gac?cta?gct?gat?gct?gct?ttg?act?gct?gct?tta?gct?gtg?gca?att?gcc????720
Asp?Leu?Ala?Asp?Ala?Ala?Leu?Thr?Ala?Ala?Leu?Ala?Val?Ala?Ile?Ala
225?????????????????230?????????????????235?????????????????240
aat?gtt?tat?ggt?gaa?aag?ccg?gtt?gat?tca?gct?tta?tct?gtc?atc?gcg????768
Asn?Val?Tyr?Gly?Glu?Lys?Pro?Val?Asp?Ser?Ala?Leu?Ser?Val?Ile?Ala
245?????????????????250?????????????????255
atg?ggc?aaa?tgt?ggc?gcg?cag?gaa?ttg?aac?tac?att?tca?gat?gtg?gac????816
Met?Gly?Lys?Cys?Gly?Ala?Gln?Glu?Leu?Asn?Tyr?Ile?Ser?Asp?Val?Asp
260?????????????????265?????????????????270
gtg?gtg?ttt?gtt?gca?gag?ccg?gca?aac?tct?aaa?tca?aca?cgc?acc?gca????864
Val?Val?Phe?Val?Ala?Glu?Pro?Ala?Asn?Ser?Lys?Ser?Thr?Arg?Thr?Ala
275?????????????????280?????????????????285
gca?gag?ctc?att?cgc?atc?ggt?agc?aac?tcg?ttc?ttt?gag?gtg?gat?gca????912
Ala?Glu?Leu?Ile?Arg?Ile?Gly?Ser?Asn?Ser?Phe?Phe?Glu?Val?Asp?Ala
290?????????????????295?????????????????300
gca?ctt?cgc?cca?gaa?ggt?aaa?agt?ggc?gct?ctt?gtg?cgc?tct?ttg?gat????960
Ala?Leu?Arg?Pro?Glu?Gly?Lys?Ser?Gly?Ala?Leu?Val?Arg?Ser?Leu?Asp
305?????????????????310?????????????????315?????????????????320
tcc?cat?atg?gcg?tat?tac?aag?cgc?tgg?gcg?gaa?acc?tgg?gaa?ttt?cag????1008
Ser?His?Met?Ala?Tyr?Tyr?Lys?Arg?Trp?Ala?Glu?Thr?Trp?Glu?Phe?Gln
325?????????????????330?????????????????335
gca?ctg?ctg?aaa?gct?cgt?ccc?atg?acg?ggt?gat?att?gac?ctt?ggg?cag????1056
Ala?Leu?Leu?Lys?Ala?Arg?Pro?Met?Thr?Gly?Asp?Ile?Asp?Leu?Gly?Gln
340?????????????????345?????????????????350
tcc?tat?gtg?gat?gct?ctt?tca?ccg?ttg?att?tgg?gcg?gct?agc?cag?cgg????1104
Ser?Tyr?Val?Asp?Ala?Leu?Ser?Pro?Leu?Ile?Trp?Ala?Ala?Ser?Gln?Arg
355?????????????????360?????????????????365
gaa?tca?ttt?gtc?aca?gat?gtc?caa?gct?atg?cgc?cgt?cga?gtg?ttg?gac????1152
Glu?Ser?Phe?Val?Thr?Asp?Val?Gln?Ala?Met?Arg?Arg?Arg?Val?Leu?Asp
370?????????????????375?????????????????380
aat?gtt?ccg?gaa?gac?ttg?cgt?gat?cgt?gag?ctg?aag?ctt?ggt?cgc?ggt????1200
Asn?Val?Pro?Glu?Asp?Leu?Arg?Asp?Arg?Glu?Leu?Lys?Leu?Gly?Arg?Gly
385?????????????????390?????????????????395?????????????????400
ggt?ttg?agg?gat?gtg?gag?ttt?gct?gtc?cag?ctc?ctt?cag?atg?gtg?cat????1248
Gly?Leu?Arg?Asp?Val?Glu?Phe?Ala?Val?Gln?Leu?Leu?Gln?Met?Val?His
405?????????????????410?????????????????415
ggt?cgc?att?gat?gag?acg?ttg?cgg?gtt?cgg?tca?acg?gta?aat?gct?ttg????1296
Gly?Arg?Ile?Asp?Glu?Thr?Leu?Arg?Val?Arg?Ser?Thr?Val?Asn?Ala?Leu
420?????????????????425?????????????????430
cat?gtg?ttg?gtt?gat?cag?gga?tat?gtg?ggt?cgt?gaa?gac?ggg?cat?aat????1344
His?Val?Leu?Val?Asp?Gln?Gly?Tyr?Val?Gly?Arg?Glu?Asp?Gly?His?Asn
435?????????????????440?????????????????445
ctc?att?gag?tcg?tat?gag?ttt?ttg?cgc?ctg?ttg?gag?cat?cgc?ctt?caa????1392
Leu?Ile?Glu?Ser?Tyr?Glu?Phe?Leu?Arg?Leu?Leu?Glu?His?Arg?Leu?Gln
450?????????????????455?????????????????460
ttg?gag?cgg?atc?aag?cgc?act?cac?ttg?tta?ccg?aaa?cct?gat?gac?cga????1440
Leu?Glu?Arg?Ile?Lys?Arg?Thr?His?Leu?Leu?Pro?Lys?Pro?Asp?Asp?Arg
465?????????????????470?????????????????475?????????????????480
atg?aat?atg?cgc?tgg?ttg?gcg?cgc?gct?tct?ggg?ttt?act?ggt?tcg?atg????1488
Met?Asn?Met?Arg?Trp?Leu?Ala?Arg?Ala?Ser?Gly?Phe?Thr?Gly?Ser?Met
485?????????????????490?????????????????495
gag?caa?agt?tcg?gcc?aaa?gct?atg?gaa?cgg?cat?ttg?cgt?aag?gtt?cgt????1536
Glu?Gln?Ser?Ser?Ala?Lys?Ala?Met?Glu?Arg?His?Leu?Arg?Lys?Val?Arg
500?????????????????505?????????????????510
ttg?cag?att?cag?tcg?ttg?cat?agt?cag?ctg?ttt?tat?cgg?cca?ctg?ctg????1584
Leu?Gln?Ile?Gln?Ser?Leu?His?Ser?Gln?Leu?Phe?Tyr?Arg?Pro?Leu?Leu
515?????????????????520?????????????????525
aac?tct?gtg?gtc?aac?ttg?agc?gcg?gat?gcc?atc?aga?ttg?tct?ccg?gat????1632
Asn?Ser?Val?Val?Asn?Leu?Ser?Ala?Asp?Ala?Ile?Arg?Leu?Ser?Pro?Asp
530?????????????????535?????????????????540
gct?gca?aag?cta?caa?ttg?ggg?gca?ttg?gga?tac?ctg?cat?cca?tca?cgt????1680
Ala?Ala?Lys?Leu?Gln?Leu?Gly?Ala?Leu?Gly?Tyr?Leu?His?Pro?Ser?Arg
545?????????????????550?????????????????555?????????????????560
gct?tat?gaa?cac?ctg?act?gct?ctt?gca?tca?gga?gct?agc?cgt?aaa?gcc????1728
Ala?Tyr?Glu?His?Leu?Thr?Ala?Leu?Ala?Ser?Gly?Ala?Ser?Arg?Lys?Ala
565?????????????????570?????????????????575
aag?att?cag?gcg?atg?ttg?ctg?ccc?acg?ttg?atg?gag?tgg?ctg?tct?caa????1776
Lys?Ile?Gln?Ala?Met?Leu?Leu?Pro?Thr?Leu?Met?Glu?Trp?Leu?Ser?Gln
580?????????????????585?????????????????590
aca?gct?gaa?cca?gat?gcg?gga?ttg?ctg?aat?tac?cgc?aag?ctt?tct?gat????1824
Thr?Ala?Glu?Pro?Asp?Ala?Gly?Leu?Leu?Asn?Tyr?Arg?Lys?Leu?Ser?Asp
595?????????????????600?????????????????605
gct?tcc?tat?gat?cgc?agc?tgg?ttt?ttg?cgc?atg?ctg?cgt?gat?gag?ggc????1872
Ala?Ser?Tyr?Asp?Arg?Ser?Trp?Phe?Leu?Arg?Met?Leu?Arg?Asp?Glu?Gly
610?????????????????615?????????????????620
gta?gtg?ggg?cag?cgg?ttg?atg?cgt?att?ttg?gga?aat?tct?ccc?tat?att????1920
Val?Val?Gly?Gln?Arg?Leu?Met?Arg?Ile?Leu?Gly?Asn?Ser?Pro?Tyr?Ile
625?????????????????630?????????????????635?????????????????640
tct?gaa?ctg?att?atc?tcc?act?ccg?gac?ttt?gtg?aaa?cag?ctg?ggt?gat????1968
Ser?Glu?Leu?Ile?Ile?Ser?Thr?Pro?Asp?Phe?Val?Lys?Gln?Leu?Gly?Asp
645?????????????????650?????????????????655
gcg?gcg?tct?ggt?cct?aaa?ttg?ctt?gct?act?gca?ccg?act?cag?gtt?gtg????2016
Ala?Ala?Ser?Gly?Pro?Lys?Leu?Leu?Ala?Thr?Ala?Pro?Thr?Gln?Val?Val
660?????????????????665?????????????????670
aaa?gca?atc?aag?gcg?acg?gtg?tcg?cgt?cat?gag?tca?cct?gat?cgg?gcg????2064
Lys?Ala?Ile?Lys?Ala?Thr?Val?Ser?Arg?His?Glu?Ser?Pro?Asp?Arg?Ala
675?????????????????680?????????????????685
atc?cag?gct?gca?cga?tcg?ctg?agg?agg?cag?gag?ctg?gca?cgc?att?gcc????2112
Ile?Gln?Ala?Ala?Arg?Ser?Leu?Arg?Arg?Gln?Glu?Leu?Ala?Arg?Ile?Ala
690?????????????????695?????????????????700
tct?gct?gat?ttg?ctc?aac?atg?ctc?act?gtt?cag?gaa?gta?tgc?caa?agc????2160
Ser?Ala?Asp?Leu?Leu?Asn?Met?Leu?Thr?Val?Gln?Glu?Val?Cys?Gln?Ser
705?????????????????710?????????????????715?????????????????720
ttg?tca?cta?gtc?tgg?gat?gcg?gtg?ttg?gat?gct?gcc?ttg?gat?gcg?gaa????2208
Leu?Ser?Leu?Val?Trp?Asp?Ala?Val?Leu?Asp?Ala?Ala?Leu?Asp?Ala?Glu
725?????????????????730?????????????????735
atc?cgt?gct?gca?ctt?aac?gat?cca?cag?aaa?cca?gat?cag?cct?ctg?gcc????2256
Ile?Arg?Ala?Ala?Leu?Asn?Asp?Pro?Gln?Lys?Pro?Asp?Gln?Pro?Leu?Ala
740?????????????????745?????????????????750
aat?att?tct?gtg?atc?ggc?atg?ggc?cgt?ttg?ggt?gga?gca?gaa?ctt?gga????2304
Asn?Ile?Ser?Val?Ile?Gly?Met?Gly?Arg?Leu?Gly?Gly?Ala?Glu?Leu?Gly
755?????????????????760?????????????????765
tac?ggt?tct?gat?gcc?gat?gtg?atg?ttt?gta?tgc?gag?ccg?gta?gcc?ggt????2352
Tyr?Gly?Ser?Asp?Ala?Asp?Val?Met?Phe?Val?Cys?Glu?Pro?Val?Ala?Gly
770?????????????????775?????????????????780
gtg?gaa?gag?cat?gag?gcc?gtc?aca?tgg?tct?att?gcg?atc?tgt?gat?tcc????2400
Val?Glu?Glu?His?Glu?Ala?Val?Thr?Trp?Ser?Ile?Ala?Ile?Cys?Asp?Ser
785?????????????????790?????????????????795?????????????????800
atg?cgg?tcg?agg?ctt?gcg?cag?cct?tcc?ggt?gat?cca?cct?ttg?gag?gtg????2448
Met?Arg?Ser?Arg?Leu?Ala?Gln?Pro?Ser?Gly?Asp?Pro?Pro?Leu?Glu?Val
805?????????????????810?????????????????815
gat?ctg?ggg?ctg?cgt?cct?gaa?ggg?aga?tct?ggt?gcg?att?gtg?cgc?acc????2496
Asp?Leu?Gly?Leu?Arg?Pro?Glu?Gly?Arg?Ser?Gly?Ala?Ile?Val?Arg?Thr
820?????????????????825?????????????????830
gtt?gat?tcc?tat?gtg?aag?tac?tac?gaa?aag?tgg?ggt?gaa?act?tgg?gag????2544
Val?Asp?Ser?Tyr?Val?Lys?Tyr?Tyr?Glu?Lys?Trp?Gly?Glu?Thr?Trp?Glu
835?????????????????840?????????????????845
att?cag?gcg?ctg?ctg?agg?gct?gcg?tgg?gtt?gct?ggt?gat?cgt?gag?ctg????2592
Ile?Gln?Ala?Leu?Leu?Arg?Ala?Ala?Trp?Val?Ala?Gly?Asp?Arg?Glu?Leu
850?????????????????855?????????????????860
ggc?att?aag?ttc?ttg?gag?tcg?att?gat?cgt?ttc?cgc?tac?cca?gtt?gac????2640
Gly?Ile?Lys?Phe?Leu?Glu?Ser?Ile?Asp?Arg?Phe?Arg?Tyr?Pro?Val?Asp
865?????????????????870?????????????????875?????????????????880
ggg?gca?acg?cag?gcg?cag?ctt?cgt?gaa?gtt?cgt?cga?att?aag?gcg?agg????2688
Gly?Ala?Thr?Gln?Ala?Gln?Leu?Arg?Glu?Val?Arg?Arg?Ile?Lys?Ala?Arg
885?????????????????890?????????????????895
gtg?gat?aat?gag?agg?ctt?ccg?cgc?ggg?gct?gat?cga?aat?acc?cat?acc????2736
Val?Asp?Asn?Glu?Arg?Leu?Pro?Arg?Gly?Ala?Asp?Arg?Asn?Thr?His?Thr
900?????????????????905?????????????????910
aag?ctg?ggt?cgg?gga?gcg?tta?act?gac?atc?gag?tgg?act?gtg?cag?ttg????2784
Lys?Leu?Gly?Arg?Gly?Ala?Leu?Thr?Asp?Ile?Glu?Trp?Thr?Val?Gln?Leu
915?????????????????920?????????????????925
ttg?acc?atg?atg?cat?gct?cat?gag?att?ccg?gag?ctg?cac?aat?acg?tcg????2832
Leu?Thr?Met?Met?His?Ala?His?Glu?Ile?Pro?Glu?Leu?His?Asn?Thr?Ser
930?????????????????935?????????????????940
acg?ttg?gaa?gtt?ctt?gaa?gtg?ctg?gaa?aag?cat?cag?att?att?aac?cct????2880
Thr?Leu?Glu?Val?Leu?Glu?Val?Leu?Glu?Lys?His?Gln?Ile?Ile?Asn?Pro
945?????????????????950?????????????????955?????????????????960
gtg?cag?gtg?cag?acg?ctt?cgg?gaa?gcg?tgg?ctg?acg?gca?acg?gct?gct????2928
Val?Gln?Val?Gln?Thr?Leu?Arg?Glu?Ala?Trp?Leu?Thr?Ala?Thr?Ala?Ala
965?????????????????970?????????????????975
agg?aat?gcg?ctt?gtg?ctg?gtc?agg?ggt?aag?aga?tta?gat?cag?tta?cct????2976
Arg?Asn?Ala?Leu?Val?Leu?Val?Arg?Gly?Lys?Arg?Leu?Asp?Gln?Leu?Pro
980?????????????????985?????????????????990
act?cct?ggt?ccg?cac?ctt?gcg?cag?gtg?gct?ggt?gcg?tct?ggt?tgg?gat????3024
Thr?Pro?Gly?Pro?His?Leu?Ala?Gln?Val?Ala?Gly?Ala?Ser?Gly?Trp?Asp
995????????????????1000????????????????1005
cca?aat?gag?tac?cag?gag?tat?ttg?gaa?aac?tat?ctg?aaa?gtg?acc?agg????3072
Pro?Asn?Glu?Tyr?Gln?Glu?Tyr?Leu?Glu?Asn?Tyr?Leu?Lys?Val?Thr?Arg
1010????????????????1015????????????????1020
aag?agt?cgt?cag?gtt?gtt?gat?gaa?gtc?ttc?tgg?ggt?gtg?gac?tct?atg????3120
Lys?Ser?Arg?Gln?Val?Val?Asp?Glu?Val?Phe?Trp?Gly?Val?Asp?Ser?Met
1025???????????????1030????????????????1035????????????????1040
gag?caa?cgt?gag?ttt?tag????????????????????????????????????????????3138
Glu?Gln?Arg?Glu?Phe
1045
<210>??18
<211>??1045
<212>??PRT
＜213〉brevibacterium
<400>??18
Met?Ser?Gly?Pro?Leu?Arg?Ser?Glu?Arg?Lys?Val?Val?Gly?Phe?Val?Arg
1???????????????5??????????????????10??????????????????15
Asp?Pro?Leu?Pro?Lys?Val?Gly?Ser?Leu?Ser?Leu?Lys?Ser?Glu?His?Ala
20??????????????????25??????????????????30
Gln?Ala?Asp?Leu?Glu?His?Leu?Gly?Trp?Arg?Asn?Val?Glu?Ser?Leu?Asp
35???????????????????40??????????????????45
Leu?Leu?Trp?Gly?Leu?Ser?Gly?Ala?Gly?Asp?Pro?Asp?Val?Ala?Leu?Asn
50??????????????????55??????????????????60
Leu?Leu?Ile?Arg?Leu?Tyr?Gln?Ala?Leu?Glu?Ala?Ile?Gly?Glu?Asp?Ala
65??????????????????70??????????????????75??????????????????80
Arg?Asn?Glu?Leu?Asp?Gln?Glu?Ile?Arg?Gln?Asp?Glu?Glu?Leu?Arg?Val
85??????????????????90??????????????????95
Arg?Leu?Phe?Ala?Leu?Leu?Gly?Gly?Ser?Ser?Ala?Val?Gly?Asp?His?Leu
100?????????????????105?????????????????110
Val?Ala?Asn?Pro?Leu?Gln?Trp?Lys?Leu?Leu?Lys?Leu?Asp?Ala?Pro?Ser
115?????????????????120?????????????????125
Arg?Glu?Glu?Met?Phe?Gln?Ala?Leu?Leu?Glu?Ser?Val?Lys?Ala?Gln?Pro
130?????????????????135?????????????????140
Ala?Val?Leu?Glu?Val?Glu?Asp?Phe?Ser?Asp?Ala?His?Asn?Ile?Ala?Arg
145?????????????????150?????????????????155?????????????????160
Asp?Asp?Leu?Ser?Thr?Pro?Gly?Phe?Tyr?Thr?Ala?Ser?Val?Thr?Gly?Pro
165?????????????????170?????????????????175
Glu?Ala?Glu?Arg?Val?Leu?Lys?Trp?Thr?Tyr?Arg?Thr?Leu?Leu?Thr?Arg
180?????????????????185?????????????????190
Ile?Ala?Ala?His?Asp?Leu?Ala?Gly?Thr?Tyr?Pro?Thr?Asp?Met?Arg?Arg
195?????????????????200?????????????????205
Lys?Gly?Gly?Asp?Pro?Val?Pro?Phe?Ser?Thr?Val?Thr?Met?Gln?Leu?Ser
210?????????????????215?????????????????220
Asp?Leu?Ala?Asp?Ala?Ala?Leu?Thr?Ala?Ala?Leu?Ala?Val?Ala?Ile?Ala
225?????????????????230?????????????????235?????????????????240
Asn?Val?Tyr?Gly?Glu?Lys?Pro?Val?Asp?Ser?Ala?Leu?Ser?Val?Ile?Ala
245?????????????????250?????????????????255
Met?Gly?Lys?Cys?Gly?Ala?Gln?Glu?Leu?Asn?Tyr?Ile?Ser?Asp?Val?Asp
260?????????????????265?????????????????270
Val?Val?Phe?Val?Ala?Glu?Pro?Ala?Asn?Ser?Lys?Ser?Thr?Arg?Thr?Ala
275?????????????????280?????????????????285
Ala?Glu?Leu?Ile?Arg?Ile?Gly?Ser?Asn?Ser?Phe?Phe?Glu?Val?Asp?Ala
290?????????????????295?????????????????300
Ala?Leu?Arg?Pro?Glu?Gly?Lys?Ser?Gly?Ala?Leu?Val?Arg?Ser?Leu?Asp
305?????????????????310?????????????????315?????????????????320
Ser?His?Met?Ala?Tyr?Tyr?Lys?Arg?Trp?Ala?Glu?Thr?Trp?Glu?Phe?Gln
325?????????????????330?????????????????335
Ala?Leu?Leu?Lys?Ala?Arg?Pro?Met?Thr?Gly?Asp?Ile?Asp?Leu?Gly?Gln
340?????????????????345?????????????????350
Ser?Tyr?Val?Asp?Ala?Leu?Ser?Pro?Leu?Ile?Trp?Ala?Ala?Ser?Gln?Arg
355?????????????????360?????????????????365
Glu?Ser?Phe?Val?Thr?Asp?Val?Gln?Ala?Met?Arg?Arg?Arg?Val?Leu?Asp
370?????????????????375?????????????????380
Asn?Val?Pro?Glu?Asp?Leu?Arg?Asp?Arg?Glu?Leu?Lys?Leu?Gly?Arg?Gly
385?????????????????390?????????????????395?????????????????400
Gly?Leu?Arg?Asp?Val?Glu?Phe?Ala?Val?Gln?Leu?Leu?Gln?Met?Val?His
405?????????????????410?????????????????415
Gly?Arg?Ile?Asp?Glu?Thr?Leu?Arg?Val?Arg?Ser?Thr?Val?Asn?Ala?Leu
420?????????????????425?????????????????430
His?Val?Leu?Val?Asp?Gln?Gly?Tyr?Val?Gly?Arg?Glu?Asp?Gly?His?Asn
435?????????????????440?????????????????445
Leu?Ile?Glu?Ser?Tyr?Glu?Phe?Leu?Arg?Leu?Leu?Glu?His?Arg?Leu?Gln
450?????????????????455?????????????????460
Leu?Glu?Arg?Ile?Lys?Arg?Thr?His?Leu?Leu?Pro?Lys?Pro?Asp?Asp?Arg
465?????????????????470?????????????????475?????????????????480
Met?Asn?Met?Arg?Trp?Leu?Ala?Arg?Ala?Ser?Gly?Phe?Thr?Gly?Ser?Met
485?????????????????490?????????????????495
Glu?Gln?Ser?Ser?Ala?Lys?Ala?Met?Glu?Arg?His?Leu?Arg?Lys?Val?Arg
500?????????????????505?????????????????510
Leu?Gln?Ile?Gln?Ser?Leu?His?Ser?Gln?Leu?Phe?Tyr?Arg?Pro?Leu?Leu
515?????????????????520?????????????????525
Asn?Ser?Val?Val?Asn?Leu?Ser?Ala?Asp?Ala?Ile?Arg?Leu?Ser?Pro?Asp
530?????????????????535?????????????????540
Ala?Ala?Lys?Leu?Gln?Leu?Gly?Ala?Leu?Gly?Tyr?Leu?His?Pro?Ser?Arg
545?????????????????550?????????????????555?????????????????560
Ala?Tyr?Glu?His?Leu?Thr?Ala?Leu?Ala?Ser?Gly?Ala?Ser?Arg?Lys?Ala
565?????????????????570?????????????????575
Lys?Ile?Gln?Ala?Met?Leu?Leu?Pro?Thr?Leu?Met?Glu?Trp?Leu?Ser?Gln
580?????????????????585?????????????????590
Thr?Ala?Glu?Pro?Asp?Ala?Gly?Leu?Leu?Asn?Tyr?Arg?Lys?Leu?Ser?Asp
595?????????????????600?????????????????605
Ala?Ser?Tyr?Asp?Arg?Ser?Trp?Phe?Leu?Arg?Met?Leu?Arg?Asp?Glu?Gly
610?????????????????615?????????????????620
Val?Val?Gly?Gln?Arg?Leu?Met?Arg?Ile?Leu?Gly?Asn?Ser?Pro?Tyr?Ile
625?????????????????630?????????????????635?????????????????640
Ser?Glu?Leu?Ile?Ile?Ser?Thr?Pro?Asp?Phe?Val?Lys?Gln?Leu?Gly?Asp
645?????????????????650?????????????????655
Ala?Ala?Ser?Gly?Pro?Lys?Leu?Leu?Ala?Thr?Ala?Pro?Thr?Gln?Val?Val
660?????????????????665?????????????????670
Lys?Ala?Ile?Lys?Ala?Thr?Val?Ser?Arg?His?Glu?Ser?Pro?Asp?Arg?Ala
675?????????????????680?????????????????685
Ile?Gln?Ala?Ala?Arg?Ser?Leu?Arg?Arg?Gln?Glu?Leu?Ala?Arg?Ile?Ala
690?????????????????695?????????????????700
Ser?Ala?Asp?Leu?Leu?Asn?Met?Leu?Thr?Val?Gln?Glu?Val?Cys?Gln?Ser
705?????????????????710?????????????????715?????????????????720
Leu?Ser?Leu?Val?Trp?Asp?Ala?Val?Leu?Asp?Ala?Ala?Leu?Asp?Ala?Glu
725?????????????????730?????????????????735
Ile?Arg?Ala?Ala?Leu?Asn?Asp?Pro?Gln?Lys?Pro?Asp?Gln?Pro?Leu?Ala
740?????????????????745?????????????????750
Asn?Ile?Ser?Val?Ile?Gly?Met?Gly?Arg?Leu?Gly?Gly?Ala?Glu?Leu?Gly
755?????????????????760?????????????????765
Tyr?Gly?Ser?Asp?Ala?Asp?Val?Met?Phe?Val?Cys?Glu?Pro?Val?Ala?Gly
770?????????????????775?????????????????780
Val?Glu?Glu?His?Glu?Ala?Val?Thr?Trp?Ser?Ile?Ala?Ile?Cys?Asp?Ser
785?????????????????790?????????????????795?????????????????800
Met?Arg?Ser?Arg?Leu?Ala?Gln?Pro?Ser?Gly?Asp?Pro?Pro?Leu?Glu?Val
805?????????????????810?????????????????815
Asp?Leu?Gly?Leu?Arg?Pro?Glu?Gly?Arg?Ser?Gly?Ala?Ile?Val?Arg?Thr
820?????????????????825?????????????????830
Val?Asp?Ser?Tyr?Val?Lys?Tyr?Tyr?Glu?Lys?Trp?Gly?Glu?Thr?Trp?Glu
835?????????????????840?????????????????845
Ile?Gln?Ala?Leu?Leu?Arg?Ala?Ala?Trp?Val?Ala?Gly?Asp?Arg?Glu?Leu
850?????????????????855?????????????????860
Gly?Ile?Lys?Phe?Leu?Glu?Ser?Ile?Asp?Arg?Phe?Arg?Tyr?Pro?Val?Asp
865?????????????????870?????????????????875?????????????????880
Gly?Ala?Thr?Gln?Ala?Gln?Leu?Arg?Glu?Val?Arg?Arg?Ile?Lys?Ala?Arg
885?????????????????890?????????????????895
Val?Asp?Asn?Glu?Arg?Leu?Pro?Arg?Gly?Ala?Asp?Arg?Asn?Thr?His?Thr
900?????????????????905?????????????????910
Lys?Leu?Gly?Arg?Gly?Ala?Leu?Thr?Asp?Ile?Glu?Trp?Thr?Val?Gln?Leu
915?????????????????920?????????????????925
Leu?Thr?Met?Met?His?Ala?His?Glu?Ile?Pro?Glu?Leu?His?Asn?Thr?Ser
930?????????????????935?????????????????940
Thr?Leu?Glu?Val?Leu?Glu?Val?Leu?Glu?Lys?His?Gln?Ile?Ile?Asn?Pro
945?????????????????950?????????????????955?????????????????960
Val?Gln?Val?Gln?Thr?Leu?Arg?Glu?Ala?Trp?Leu?Thr?Ala?Thr?Ala?Ala
965?????????????????970?????????????????975
Arg?Asn?Ala?Leu?Val?Leu?Val?Arg?Gly?Lys?Arg?Leu?Asp?Gln?Leu?Pro
980?????????????????985?????????????????990
Thr?Pro?Gly?Pro?His?Leu?Ala?Gln?Val?Ala?Gly?Ala?Ser?Gly?Trp?Asp
995????????????????1000????????????????1005
Pro?Asn?Glu?Tyr?Gln?Glu?Tyr?Leu?Glu?Asn?Tyr?Leu?Lys?Val?Thr?Arg
1010????????????????1015????????????????1020
Lys?Ser?Arg?Gln?Val?Val?Asp?Glu?Val?Phe?Trp?Gly?Val?Asp?Ser?Met
1025???????????????1030????????????????1035????????????????1040
Glu?Gln?Arg?Glu?Phe
1045
<210>??19
<211>??1434
<212>??DNA
＜213〉brevibacterium
<220>
<221>??CDS
<222>??(1)..(1434)
<400>??19
gtg?gcg?ttt?gaa?acc?ccg?gaa?gaa?att?gtc?aag?ttc?atc?aag?gat?gaa????48
Val?Ala?Phe?Glu?Thr?Pro?Glu?Glu?Ile?Val?Lys?Phe?Ile?Lys?Asp?Glu
1???????????????5??????????????????10??????????????????15
aac?gtc?gag?ttc?gtt?gac?gtt?cga?ttc?acc?gac?ctt?ccc?ggc?acc?gag????96
Asn?Val?Glu?Phe?Val?Asp?Val?Arg?Phe?Thr?Asp?Leu?Pro?Gly?Thr?Glu
20??????????????????25??????????????????30
cag?cac?ttc?agc?atc?cca?gct?gcc?agc?ttc?gat?gca?gat?aca?gtc?gaa????144
Gln?His?Phe?Ser?Ile?Pro?Ala?Ala?Ser?Phe?Asp?Ala?Asp?Thr?Val?Glu
35??????????????????40??????????????????45
gaa?ggt?ctc?gca?ttc?gac?gga?tcc?tcg?atc?cgt?ggc?ttc?acc?acg?atc????192
Glu?Gly?Leu?Ala?Phe?Asp?Gly?Ser?Ser?Ile?Arg?Gly?Phe?Thr?Thr?Ile
50???????????????????55??????????????????60
gac?gaa?tct?gac?atg?aat?ctc?ctg?cca?gac?ctc?gga?acg?gcc?acc?ctt????240
Asp?Glu?Ser?Asp?Met?Asn?Leu?Leu?Pro?Asp?Leu?Gly?Thr?Ala?Thr?Leu
65??????????????????70??????????????????75??????????????????80
gat?cca?ttc?cgc?aag?gca?aag?acc?ctg?aac?gtt?aag?ttc?ttc?gtt?cac????288
Asp?Pro?Phe?Arg?Lys?Ala?Lys?Thr?Leu?Asn?Val?Lys?Phe?Phe?Val?His
85??????????????????90??????????????????95
gat?cct?ttc?acc?cgc?gag?gca?ttc?tcc?cgc?gac?cca?cgc?aac?gta?gca????336
Asp?Pro?Phe?Thr?Arg?Glu?Ala?Phe?Ser?Arg?Asp?Pro?Arg?Asn?Val?Ala
100?????????????????105?????????????????110
cgc?aag?gca?gag?cag?tac?ctg?gca?tcc?acc?ggc?att?gca?gac?acc?tgc????384
Arg?Lys?Ala?Glu?Gln?Tyr?Leu?Ala?Ser?Thr?Gly?Ile?Ala?Asp?Thr?Cys
115?????????????????120?????????????????125
aac?ttc?ggc?gcc?gag?gct?gag?ttc?tac?ctc?ttc?gac?tcc?gtt?cgc?tac????432
Asn?Phe?Gly?Ala?Glu?Ala?Glu?Phe?Tyr?Leu?Phe?Asp?Ser?Val?Arg?Tyr
130?????????????????135?????????????????140
tcc?acc?gag?atg?aac?tcc?ggc?ttc?tac?gaa?gta?gat?acc?gaa?gaa?ggc????480
Ser?Thr?Glu?Met?Asn?Ser?Gly?Phe?Tyr?Glu?Val?Asp?Thr?Glu?Glu?Gly
145?????????????????150?????????????????155?????????????????160
tgg?tgg?aac?cgt?ggc?aag?gaa?acc?aac?ctc?gac?gga?acc?cca?aac?ctg????528
Trp?Trp?Asn?Arg?Gly?Lys?Glu?Thr?Asn?Leu?Asp?Gly?Thr?Pro?Asn?Leu
165?????????????????170?????????????????175
ggc?gca?aag?aac?cgc?gtc?aag?ggt?ggc?tac?ttc?cca?gta?gca?cca?tac????576
Gly?Ala?Lys?Asn?Arg?Val?Lys?Gly?Gly?Tyr?Phe?Pro?Val?Ala?Pro?Tyr
180?????????????????185?????????????????190
gac?caa?acc?gtt?gac?gtg?cgc?gat?gac?atg?gtt?cgc?aac?ctc?gca?gct????624
Asp?Gln?Thr?Val?Asp?Val?Arg?Asp?Asp?Met?Val?Arg?Asn?Leu?Ala?Ala
195?????????????????200?????????????????205
tcc?ggc?ttc?gct?ctt?gag?cgt?ttc?cac?cac?gaa?gtc?ggt?ggc?gga?cag????672
Ser?Gly?Phe?Ala?Leu?Glu?Arg?Phe?His?His?Glu?Val?Gly?Gly?Gly?Gln
210?????????????????215?????????????????220
cag?gaa?atc?aac?tac?cgc?ttc?aac?acc?atg?ctc?cac?gcg?gca?gat?gat????720
Gln?Glu?Ile?Asn?Tyr?Arg?Phe?Asn?Thr?Met?Leu?His?Ala?Ala?Asp?Asp
225?????????????????230?????????????????235?????????????????240
atc?cag?acc?ttc?aag?tac?atc?atc?aag?aac?acc?gct?cgc?ctc?cac?ggc????768
Ile?Gln?Thr?Phe?Lys?Tyr?Ile?Ile?Lys?Asn?Thr?Ala?Arg?Leu?His?Gly
245?????????????????250?????????????????255
aag?gct?gca?acc?ttc?atg?cct?aag?cca?ctg?gct?ggc?gac?aac?ggt?tcc????816
Lys?Ala?Ala?Thr?Phe?Met?Pro?Lys?Pro?Leu?Ala?Gly?Asp?Asn?Gly?Ser
260?????????????????265?????????????????270
ggc?atg?cac?gct?cac?cag?tcc?ctc?tgg?aag?gac?ggc?aag?cca?ctc?ttc????864
Gly?Met?His?Ala?His?Gln?Ser?Leu?Trp?Lys?Asp?Gly?Lys?Pro?Leu?Phe
275?????????????????280?????????????????285
cac?gat?gag?tcc?ggc?tac?gca?ggc?ctg?tcc?gac?atc?gcc?cgc?tac?tac????912
His?Asp?Glu?Ser?Gly?Tyr?Ala?Gly?Leu?Ser?Asp?Ile?Ala?Arg?Tyr?Tyr
290?????????????????295?????????????????300
atc?ggc?ggc?atc?ctg?cac?cac?gca?ggc?gct?gtt?ctg?gcg?ttc?acc?aac????960
Ile?Gly?Gly?Ile?Leu?His?His?Ala?Gly?Ala?Val?Leu?Ala?Phe?Thr?Asn
305?????????????????310?????????????????315?????????????????320
gca?acc?ctg?aac?tcc?tac?cac?cgt?ctg?gtt?cca?ggc?ttc?gag?gct?cca????1008
Ala?Thr?Leu?Asn?Ser?Tyr?His?Arg?Leu?Val?Pro?Gly?Phe?Glu?Ala?Pro
325?????????????????330?????????????????335
atc?aac?ctg?gtg?tac?tca?cag?cgc?aac?cgt?tcc?gct?gct?gtc?cgt?atc????1056
Ile?Asn?Leu?Val?Tyr?Ser?Gln?Arg?Asn?Arg?Ser?Ala?Ala?Val?Arg?Ile
340?????????????????345?????????????????350
cca?atc?acc?gga?tcc?aac?cca?aag?gca?aag?cgc?atc?gaa?ttc?cgc?gct????1104
Pro?Ile?Thr?Gly?Ser?Asn?Pro?Lys?Ala?Lys?Arg?Ile?Glu?Phe?Arg?Ala
355?????????????????360?????????????????365
cca?gac?cca?tca?ggc?aac?cca?tac?ctg?ggc?ttc?gca?gcg?atg?atg?atg????1152
Pro?Asp?Pro?Ser?Gly?Asn?Pro?Tyr?Leu?Gly?Phe?Ala?Ala?Met?Met?Met
370?????????????????375?????????????????380
gcc?ggc?ctc?gac?ggc?atc?aag?aac?cgc?atc?gag?cca?cac?gct?cca?gtg????1200
Ala?Gly?Leu?Asp?Gly?Ile?Lys?Asn?Arg?Ile?Glu?Pro?His?Ala?Pro?Val
385?????????????????390?????????????????395?????????????????400
gac?aag?gac?ctc?tac?gaa?ctg?cca?cca?gag?gaa?gct?gca?tcc?att?cca????1248
Asp?Lys?Asp?Leu?Tyr?Glu?Leu?Pro?Pro?Glu?Glu?Ala?Ala?Ser?Ile?Pro
405?????????????????410?????????????????415
cag?gca?cca?acc?tcc?ctg?gaa?gca?tcc?ctg?aag?gca?ctg?cag?gaa?gac????1296
Gln?Ala?Pro?Thr?Ser?Leu?Glu?Ala?Ser?Leu?Lys?Ala?Leu?Gln?Glu?Asp
420?????????????????425?????????????????430
acc?gac?ttc?ctc?acc?gag?tct?gac?gtc?ttc?acc?gag?gat?ctc?atc?gag????1344
Thr?Asp?Phe?Leu?Thr?Glu?Ser?Asp?Val?Phe?Thr?Glu?Asp?Leu?Ile?Glu
435?????????????????440?????????????????445
gcg?tac?atc?cag?tac?aag?tac?gac?aac?gag?atc?tcc?cca?gtt?cgc?ctg????1392
Ala?Tyr?Ile?Gln?Tyr?Lys?Tyr?Asp?Asn?Glu?Ile?Ser?Pro?Val?Arg?Leu
450?????????????????455?????????????????460
cgc?cca?acc?ccg?cag?gaa?ttc?gaa?ttg?tac?ttc?gac?tgc?taa????????????1434
Arg?Pro?Thr?Pro?Gln?Glu?Phe?Glu?Leu?Tyr?Phe?Asp?Cys
465?????????????????470?????????????????475
<210>??20
<211>??477
<212>??PRT
＜213〉brevibacterium
<400>??20
Val?Ala?Phe?Glu?Thr?Pro?Glu?Glu?Ile?Val?Lys?Phe?Ile?Lys?Asp?Glu
1??????????????5???????????????????10??????????????????15
Asn?Val?Glu?Phe?Val?Asp?Val?Arg?Phe?Thr?Asp?Leu?Pro?Gly?Thr?Glu
20??????????????????25??????????????????30
Gln?His?Phe?Ser?Ile?Pro?Ala?Ala?Ser?Phe?Asp?Ala?Asp?Thr?Val?Glu
35??????????????????40??????????????????45
Glu?Gly?Leu?Ala?Phe?Asp?Gly?Ser?Ser?Ile?Arg?Gly?Phe?Thr?Thr?Ile
50???????????????????55??????????????????60
Asp?Glu?Ser?Asp?Met?Asn?Leu?Leu?Pro?Asp?Leu?Gly?Thr?Ala?Thr?Leu
65??????????????????70??????????????????75??????????????????80
Asp?Pro?Phe?Arg?Lys?Ala?Lys?Thr?Leu?Asn?Val?Lys?Phe?Phe?Val?His
85??????????????????90??????????????????95
Asp?Pro?Phe?Thr?Arg?Glu?Ala?Phe?Ser?Arg?Asp?Pro?Arg?Asn?Val?Ala
100?????????????????105?????????????????110
Arg?Lys?Ala?Glu?Gln?Tyr?Leu?Ala?Ser?Thr?Gly?Ile?Ala?Asp?Thr?Cys
115?????????????????120?????????????????125
Asn?Phe?Gly?Ala?Glu?Ala?Glu?Phe?Tyr?Leu?Phe?Asp?Ser?Val?Arg?Tyr
130?????????????????135?????????????????140
Ser?Thr?Glu?Met?Asn?Ser?Gly?Phe?Tyr?Glu?Val?Asp?Thr?Glu?Glu?Gly
145?????????????????150?????????????????155?????????????????160
Trp?Trp?Asn?Arg?Gly?Lys?Glu?Thr?Asn?Leu?Asp?Gly?Thr?Pro?Asn?Leu
165?????????????????170?????????????????175
Gly?Ala?Lys?Asn?Arg?Val?Lys?Gly?Gly?Tyr?Phe?Pro?Val?Ala?Pro?Tyr
180?????????????????185?????????????????190
Asp?Gln?Thr?Val?Asp?Val?Arg?Asp?Asp?Met?Val?Arg?Asn?Leu?Ala?Ala
195?????????????????200?????????????????205
Ser?Gly?PheAla?Leu?Glu?Arg?Phe?His?His?Glu?Val?Gly?Gly?Gly?Gln
210????????????????215?????????????????220
Gln?Glu?Ile?Asn?Tyr?Arg?Phe?Asn?Thr?Met?Leu?His?Ala?Ala?Asp?Asp
225?????????????????230?????????????????235?????????????????240
Ile?Gln?Thr?Phe?Lys?Tyr?Ile?Ile?Lys?Asn?Thr?Ala?Arg?Leu?His?Gly
245?????????????????250?????????????????255
Lys?Ala?Ala?Thr?Phe?Met?Pro?Lys?Pro?Leu?Ala?Gly?Asp?Asn?Gly?Ser
260?????????????????265?????????????????270
Gly?Met?His?Ala?His?Gln?Ser?Leu?Trp?Lys?Asp?Gly?Lys?Pro?Leu?Phe
275?????????????????280?????????????????285
His?Asp?Glu?Ser?Gly?Tyr?Ala?Gly?Leu?Ser?Asp?Ile?Ala?Arg?Tyr?Tyr
290?????????????????295?????????????????300
Ile?Gly?Gly?Ile?Leu?His?His?Ala?Gly?Ala?Val?Leu?Ala?Phe?Thr?Asn
305?????????????????310?????????????????315?????????????????320
Ala?Thr?Leu?Asn?Ser?Tyr?His?Arg?Leu?Val?Pro?Gly?Phe?Glu?Ala?Pro
325?????????????????330?????????????????335
Ile?Asn?Leu?Val?Tyr?Ser?Gln?Arg?Asn?Arg?Ser?Ala?Ala?Val?Arg?Ile
340?????????????????345?????????????????350
Pro?Ile?Thr?Gly?Ser?Asn?Pro?Lys?Ala?Lys?Arg?Ile?Glu?Phe?Arg?Ala
355?????????????????360?????????????????365
Pro?Asp?Pro?Ser?Gly?Asn?Pro?Tyr?Leu?Gly?Phe?Ala?Ala?Met?Met?Met
370?????????????????375?????????????????380
Ala?Gly?Leu?Asp?Gly?Ile?Lys?Asn?Arg?Ile?Glu?Pro?His?Ala?Pro?Val
385?????????????????390?????????????????395?????????????????400
Asp?Lys?Asp?Leu?Tyr?Glu?Leu?Pro?Pro?Glu?Glu?Ala?Ala?Ser?Ile?Pro
405?????????????????410?????????????????415
Gln?Ala?Pro?Thr?Ser?Leu?Glu?Ala?Ser?Leu?Lys?Ala?Leu?Gln?Glu?Asp
420?????????????????425?????????????????430
Thr?Asp?Phe?Leu?Thr?Glu?Ser?Asp?Val?Phe?Thr?Glu?Asp?Leu?Ile?Glu
435?????????????????440?????????????????445
Ala?Tyr?Ile?Gln?Tyr?Lys?Tyr?Asp?Asn?Glu?Ile?Ser?Pro?Val?Arg?Leu
450?????????????????455?????????????????460
Arg?Pro?Thr?Pro?Gln?Glu?Phe?Glu?Leu?Tyr?Phe?Asp?Cys
465?????????????????470?????????????????475
<210>??21
<211>??672
<212>??DNA
＜213〉brevibacterium
<220>
<221>??CDS
<222>??(1)..(669)
<400>??21
atg?gca?gga?gca?gtg?gga?cgc?ccc?cgg?aga?tca?gct?ccg?cga?cgg?gca????48
Met?Ala?Gly?Ala?Val?Gly?Arg?Pro?Arg?Arg?Ser?Ala?Pro?Arg?Arg?Ala
1???????????????5??????????????????10??????????????????15
ggc?aag?aat?cct?cgc?gag?gag?att?ctt?gac?gcc?tct?gct?gag?ctt?ttc????96
Gly?Lys?Asn?Pro?Arg?Glu?Glu?Ile?Leu?Asp?Ala?Ser?Ala?Glu?Leu?Phe
20??????????????????25??????????????????30
acc?cat?caa?ggc?ttc?gca?aca?acc?tcc?acg?cat?caa?atc?gct?gat?gcc????144
Thr?His?Gln?Gly?Phe?Ala?Thr?Thr?Ser?Thr?His?Gln?Ile?Ala?Asp?Ala
35??????????????????40??????????????????45
gtg?gga?atc?cgc?caa?gcc?tcg?ctg?tat?tat?cac?ttc?ccg?tct?aag?acg????192
Val?Gly?Ile?Arg?Gln?Ala?Ser?Leu?Tyr?Tyr?His?Phe?Pro?Ser?Lys?Thr
50??????????????????55??????????????????60
gaa?atc?ttc?ctc?acc?ctc?ctg?aaa?tct?acc?gtc?gag?ccg?tcc?act?gtg????240
Glu?Ile?Phe?Leu?Thr?Leu?Leu?Lys?Ser?Thr?Val?Glu?Pro?Ser?Thr?Val
65??????????????????70??????????????????75??????????????????80
ctc?gcc?gaa?gac?tta?agc?atc?ctg?gat?gca?gga?cct?gag?atg?cgc?ctc????288
Leu?Ala?Glu?Asp?Leu?Ser?Ile?Leu?Asp?Ala?Gly?Pro?Glu?Met?Arg?Leu
85??????????????????90??????????????????95
tgg?gca?atc?gtt?gcc?tcc?gaa?gtg?cgt?ctg?ctg?ctg?tcc?acc?aag?tgg????336
Trp?Ala?Ile?Val?Ala?Ser?Glu?Val?Arg?Leu?Leu?Leu?Ser?Thr?Lys?Trp
100?????????????????105?????????????????110
aac?gtc?ggt?cgc?ctg?tac?caa?ctc?ccc?atc?gtt?ggt?tct?gaa?gag?ttc????384
Asn?Val?Gly?Arg?Leu?Tyr?Gln?Leu?Pro?Ile?Val?Gly?Ser?Glu?Glu?Phe
115?????????????????120?????????????????125
gcc?gag?tac?cac?agc?cag?cgc?gaa?gcc?ctc?acc?aac?atc?ttc?cgc?gac????432
Ala?Glu?Tyr?His?Ser?Gln?Arg?Glu?Ala?Leu?Thr?Asn?Ile?Phe?Arg?Asp
130?????????????????135?????????????????140
ctc?gcc?acc?gaa?atc?gtc?ggt?gac?gac?ccc?cgc?gca?gaa?ctc?ccc?ttc????480
Leu?Ala?Thr?Glu?Ile?Val?Gly?Asp?Asp?Pro?Arg?Ala?Glu?Leu?Pro?Phe
145?????????????????150?????????????????155?????????????????160
cac?atc?acc?atg?tcg?gtg?atc?gaa?atg?cgt?cgc?aac?gac?ggc?aag?att????528
His?Ile?Thr?Met?Ser?Val?Ile?Glu?Met?Arg?Arg?Asn?Asp?Gly?Lys?Ile
165?????????????????170?????????????????175
cca?agc?ccg?ctt?tcc?gca?gac?agc?ctc?ccg?gag?acc?gca?att?atg?ctt????576
Pro?Ser?Pro?Leu?Ser?Ala?Asp?Ser?Leu?Pro?Glu?Thr?Ala?Ile?Met?Leu
180?????????????????185?????????????????190
gcc?gac?gcc?tcc?ctc?gcc?gtc?ctc?ggc?gcg?tcg?ctg?ccc?gcc?gac?cgg????624
Ala?Asp?Ala?Ser?Leu?Ala?Val?Leu?Gly?Ala?Ser?Leu?Pro?Ala?Asp?Arg
195?????????????????200?????????????????205
gtc?gaa?aaa?acg?ctt?gaa?cta?atc?aag?cag?gct?gac?gcg?aaa?taa?cca????672
Val?Glu?Lys?Thr?Leu?Glu?Leu?Ile?Lys?Gln?Ala?Asp?Ala?Lys
210?????????????????215?????????????????220
<210>??22
<211>??222
<212>??PRT
＜213〉brevibacterium
<400>??22
Met?Ala?Gly?Ala?Val?Gly?Arg?Pro?Arg?Arg?Ser?Ala?Pro?Arg?Arg?Ala
1??????????????5???????????????????10?????????????????15
Gly?Lys?Asn?Pro?Arg?Glu?Glu?Ile?Leu?Asp?Ala?Ser?Ala?Glu?Leu?Phe
20??????????????????25??????????????????30
Thr?His?Gln?Gly?Phe?Ala?Thr?Thr?Ser?Thr?His?Gln?Ile?Ala?Asp?Ala
35??????????????????40??????????????????45
Val?Gly?Ile?Arg?Gln?Ala?Ser?Leu?Tyr?Tyr?His?Phe?Pro?Ser?Lys?Thr
50??????????????????55??????????????????60
Glu?Ile?Phe?Leu?Thr?Leu?Leu?Lys?Ser?Thr?Val?Glu?Pro?Ser?Thr?Val
65??????????????????70??????????????????75??????????????????80
Leu?Ala?Glu?Asp?Leu?Ser?Ile?Leu?Asp?Ala?Gly?Pro?Glu?Met?Arg?Leu
85??????????????????90??????????????????95
Trp?Ala?Ile?Val?Ala?Ser?Glu?Val?Arg?Leu?Leu?Leu?Ser?Thr?Lys?Trp
100?????????????????105?????????????????110
Asn?Val?Gly?Arg?Leu?Tyr?Gln?Leu?Pro?Ile?Val?Gly?Ser?Glu?Glu?Phe
115?????????????????120?????????????????125
Ala?Glu?Tyr?His?Ser?Gln?Arg?Glu?Ala?Leu?Thr?Asn?Ile?Phe?Arg?Asp
130?????????????????135?????????????????140
Leu?Ala?Thr?Glu?Ile?Val?Gly?Asp?Asp?Pro?Arg?Ala?Glu?Leu?Pro?Phe
145?????????????????150?????????????????155?????????????????160
His?Ile?Thr?Met?Ser?Val?Ile?Glu?Met?Arg?Arg?Asn?Asp?Gly?Lys?Ile
165?????????????????170?????????????????175
Pro?Ser?Pro?Leu?Ser?Ala?Asp?Ser?Leu?Pro?Glu?Thr?Ala?Ile?Met?Leu
180?????????????????185?????????????????190
Ala?Asp?Ala?Ser?Leu?Ala?Val?Leu?Gly?Ala?Ser?Leu?Pro?Ala?Asp?Arg
195?????????????????200?????????????????205
Val?Glu?Lys?Thr?Leu?Glu?Leu?Ile?Lys?Gln?Ala?Asp?Ala?Lys
210?????????????????215?????????????????220
<210>??23
<211>??2076
<212>??DNA
＜213〉brevibacterium
<220>
<221>??CDS
<222>??(1)..(2076)
<400>??23
atg?aat?aat?cca?gcc?cag?ctg?cgc?caa?gat?act?gaa?aag?gaa?gtc?ctg????48
Met?Asn?Asn?Pro?Ala?Gln?Leu?Arg?Gln?Asp?Thr?Glu?Lys?Glu?Val?Leu
1???????????????5??????????????????10??????????????????15
gcg?ttg?ctg?ggc?tct?ttg?gtt?tta?ccc?gcc?ggc?acc?gcg?ctt?gcc?gcc????96
Ala?Leu?Leu?Gly?Ser?Leu?Val?Leu?Pro?Ala?Gly?Thr?Ala?Leu?Ala?Ala
20??????????????????25??????????????????30
acc?gga?tct?ttg?gcc?agg?tcc?gaa?ctc?acg?ccg?tat?tcc?gat?ttg?gac????144
Thr?Gly?Ser?Leu?Ala?Arg?Ser?Glu?Leu?Thr?Pro?Tyr?Ser?Asp?Leu?Asp
35??????????????????40??????????????????45
ctc?att?ttg?atc?cat?cca?cca?ggg?gca?acc?ccg?gat?ggc?gtg?gag?gat????192
Leu?Ile?Leu?Ile?His?Pro?Pro?Gly?Ala?Thr?Pro?Asp?Gly?Val?Glu?Asp
50??????????????????55??????????????????60
ttg?tgg?tac?ccg?att?tgg?gac?gca?aaa?aag?cgc?ctc?gac?tac?tcc?gtg????240
Leu?Trp?Tyr?Pro?Ile?Trp?Asp?Ala?Lys?Lys?Arg?Leu?Asp?Tyr?Ser?Val
65??????????????????70??????????????????75??????????????????80
cgc?acc?cca?gat?gag?tgc?gtg?gct?atg?att?tct?gcg?gat?tcc?act?gca????288
Arg?Thr?Pro?Asp?Glu?Cys?Val?Ala?Met?Ile?Ser?Ala?Asp?Ser?Thr?Ala
85??????????????????90??????????????????95
gcc?ctt?gcc?atg?ctt?gac?ctg?cga?ttt?att?gct?ggc?gat?gag?gat?ctg????336
Ala?Leu?Ala?Met?Leu?Asp?Leu?Arg?Phe?Ile?Ala?Gly?Asp?Glu?Asp?Leu
100?????????????????105?????????????????110
tgt?gcc?aaa?acg?cgc?cgg?cgc?atc?gtg?gag?aag?tgg?cgc?cag?gaa?ctc????384
Cys?Ala?Lys?Thr?Arg?Arg?Arg?Ile?Val?Glu?Lys?Trp?Arg?Gln?Glu?Leu
115?????????????????120?????????????????125
aac?aaa?aac?ttc?gac?gcc?gtt?gtg?gac?acc?gcg?att?gcc?cgt?tgg?cgc????432
Asn?Lys?Asn?Phe?Asp?Ala?Val?Val?Asp?Thr?Ala?Ile?Ala?Arg?Trp?Arg
130?????????????????135?????????????????140
cgc?tcc?gga?ccc?gtc?gtg?gca?atg?acg?cgg?cca?gat?ctt?aaa?cac?ggc????480
Arg?Ser?Gly?Pro?Val?Val?Ala?Met?Thr?Arg?Pro?Asp?Leu?Lys?His?Gly
145?????????????????150?????????????????155?????????????????160
agg?gga?ggg?ctg?cgc?gat?ttc?gaa?ctg?atc?aag?gcc?ctc?gcg?ctc?ggc????528
Arg?Gly?Gly?Leu?Arg?Asp?Phe?Glu?Leu?Ile?Lys?Ala?Leu?Ala?Leu?Gly
165?????????????????170?????????????????175
cac?cta?tgc?aac?gtt?cca?cag?cta?gat?acg?caa?cac?cag?ctg?ctt?ctc????576
His?Leu?Cys?Asn?Val?Pro?Gln?Leu?Asp?Thr?Gln?His?Gln?Leu?Leu?Leu
180?????????????????185?????????????????190
gac?gcc?cgc?acc?ttg?ctg?cac?gtc?cac?gcg?cga?cgc?tcc?cgc?gac?gtc????624
Asp?Ala?Arg?Thr?Leu?Leu?His?Val?His?Ala?Arg?Arg?Ser?Arg?Asp?Val
195?????????????????200?????????????????205
ctt?gat?ccc?gaa?ttt?gcg?gtg?gat?gtg?gcc?atg?gat?ttg?ggc?ttt?gtt????672
Leu?Asp?Pro?Glu?Phe?Ala?Val?Asp?Val?Ala?Met?Asp?Leu?Gly?Phe?Val
210?????????????????215?????????????????220
gac?cgc?tat?cac?tta?ggc?cgg?gag?atc?gcc?gat?gca?gcc?cgc?gcc?att????720
Asp?Arg?Tyr?His?Leu?Gly?Arg?Glu?Ile?Ala?Asp?Ala?Ala?Arg?Ala?Ile
225?????????????????230?????????????????235?????????????????240
gat?gac?ggc?ctg?acc?acc?gcg?ctg?gcc?acc?gcc?cgt?ggc?att?ttg?cca????768
Asp?Asp?Gly?Leu?Thr?Thr?Ala?Leu?Ala?Thr?Ala?Arg?Gly?Ile?Leu?Pro
245?????????????????250?????????????????255
cgt?cgc?acg?ggt?ttt?gct?ttt?agg?aat?gct?tct?cga?cgc?cca?ctt?gat????816
Arg?Arg?Thr?Gly?Phe?Ala?Phe?Arg?Asn?Ala?Ser?Arg?Arg?Pro?Leu?Asp
260?????????????????265?????????????????270
ctt?gat?gtc?gtc?gac?gcc?aac?ggc?act?atc?gaa?ttg?tcc?aaa?aaa?cca????864
Leu?Asp?Val?Val?Asp?Ala?Asn?Gly?Thr?Ile?Glu?Leu?Ser?Lys?Lys?Pro
275?????????????????280?????????????????285
gat?ctt?aat?gat?ccc?gca?ctt?cca?ctt?cga?gtg?gcc?gca?gcc?gca?gcg????912
Asp?Leu?Asn?Asp?Pro?Ala?Leu?Pro?Leu?Arg?Val?Ala?Ala?Ala?Ala?Ala
290?????????????????295?????????????????300
acc?acc?gga?ctt?ccg?gtg?gca?gaa?tca?acc?tgg?gct?cga?ctt?aat?gaa????960
Thr?Thr?Gly?Leu?Pro?Val?Ala?Glu?Ser?Thr?Trp?Ala?Arg?Leu?Asn?Glu
305?????????????????310?????????????????315?????????????????320
tgc?ccg?cca?ctt?cct?gag?cca?tgg?cct?gcc?aat?gca?gca?ggg?gac?ttc????1008
Cys?Pro?Pro?Leu?Pro?Glu?Pro?Trp?Pro?Ala?Asn?Ala?Ala?Gly?Asp?Phe
325?????????????????330?????????????????335
ttt?cgg?att?ctc?tcc?agt?ccg?aaa?aac?tca?cgc?cga?gtg?gtg?aaa?aat????1056
Phe?Arg?Ile?Leu?Ser?Ser?Pro?Lys?Asn?Ser?Arg?Arg?Val?Val?Lys?Asn
340?????????????????345?????????????????350
atg?gat?cgc?cac?gga?ttg?tgg?tcg?cgt?ttt?gtt?cca?gaa?tgg?gac?cgc????1104
Met?Asp?Arg?His?Gly?Leu?Trp?Ser?Arg?Phe?Val?Pro?Glu?Trp?Asp?Arg
355?????????????????360?????????????????365
atc?aaa?ggg?ctt?atg?ccc?cgt?gaa?ccc?agc?cat?att?tcc?acc?atc?gat????1152
Ile?Lys?Gly?Leu?Met?Pro?Arg?Glu?Pro?Ser?His?Ile?Ser?Thr?Ile?Asp
370?????????????????375?????????????????380
gaa?cat?agt?ctg?aac?act?gtt?gca?gga?tgt?gcg?cta?gaa?act?gtg?acc????1200
Glu?His?Ser?Leu?Asn?Thr?Val?Ala?Gly?Cys?Ala?Leu?Glu?Thr?Val?Thr
385?????????????????390?????????????????395?????????????????400
gtc?gcg?cgc?ccc?gat?ctt?tta?gtt?ttg?gga?gcc?ttg?tac?cac?gac?att????1248
Val?Ala?Arg?Pro?Asp?Leu?Leu?Val?Leu?Gly?Ala?Leu?Tyr?His?Asp?Ile
405?????????????????410?????????????????415
ggc?aag?ggc?ttc?ccg?cgt?cca?cac?gaa?caa?gta?ggt?gca?gag?atg?gtg????1296
Gly?Lys?Gly?Phe?Pro?Arg?Pro?His?Glu?Gln?Val?Gly?Ala?Glu?Met?Val
420?????????????????425?????????????????430
gcg?agg?gcc?gcg?agc?cgc?atg?ggg?ttg?aac?ctt?cgc?gat?cgt?gcc?agc????1344
Ala?Arg?Ala?Ala?Ser?Arg?Met?Gly?Leu?Asn?Leu?Arg?Asp?Arg?Ala?Ser
435?????????????????440?????????????????445
gtg?caa?acg?ctg?gtc?gcc?gag?cac?acc?gcg?gtg?gcc?aaa?atc?gcc?gcg????1392
Val?Gln?Thr?Leu?Val?Ala?Glu?His?Thr?Ala?Val?Ala?Lys?Ile?Ala?Ala
450?????????????????455?????????????????460
cgc?ctt?gat?ccc?tcc?tcg?gag?ggc?gcc?gtc?gat?aag?ctg?ctt?gat?gct????1440
Arg?Leu?Asp?Pro?Ser?Ser?Glu?Gly?Ala?Val?Asp?Lys?Leu?Leu?Asp?Ala
465?????????????????470?????????????????475?????????????????480
gtt?agg?tat?gac?ctg?gtg?aca?ttg?aat?ctg?ctt?gag?gtg?cta?aca?gaa????1488
Val?Arg?Tyr?Asp?Leu?Val?Thr?Leu?Asn?Leu?Leu?Glu?Val?Leu?Thr?Glu
485?????????????????490?????????????????495
gct?gat?gcg?aaa?gcc?acg?ggg?cct?ggc?gta?tgg?acg?gcg?cgt?ttg?gag????1536
Ala?Asp?Ala?Lys?Ala?Thr?Gly?Pro?Gly?Val?Trp?Thr?Ala?Arg?Leu?Glu
500?????????????????505?????????????????510
cat?gcg?ctg?cgg?att?gtg?tgc?aag?cgt?gcg?cgt?gat?cgc?ctc?acc?gat????1584
His?Ala?Leu?Arg?Ile?Val?Cys?Lys?Arg?Ala?Arg?Asp?Arg?Leu?Thr?Asp
515?????????????????520?????????????????525
att?cgc?ccg?gtt?gcg?ccg?atg?att?gcg?ccg?cgt?agc?gaa?att?ggt?ttg????1632
Ile?Arg?Pro?Val?Ala?Pro?Met?Ile?Ala?Pro?Arg?Ser?Glu?Ile?Gly?Leu
530?????????????????535?????????????????540
gtg?gaa?cgc?gat?ggc?gtg?ttc?aca?gtg?caa?tgg?cac?ggc?gaa?gac?tta????1680
Val?Glu?Arg?Asp?Gly?Val?Phe?Thr?Val?Gln?Trp?His?Gly?Glu?Asp?Leu
545?????????????????550?????????????????555?????????????????560
cat?cgg?att?ctt?ggc?gta?att?tat?gcc?aaa?gga?tgg?aca?atc?acc?gcg????1728
His?Arg?Ile?Leu?Gly?Val?Ile?Tyr?Ala?Lys?Gly?Trp?Thr?Ile?Thr?Ala
565?????????????????570?????????????????575
gcg?cgc?atg?ctg?gcc?aat?ggt?caa?tgg?agt?gcg?gaa?ttt?gat?gtc?cgc????1776
Ala?Arg?Met?Leu?Ala?Asn?Gly?Gln?Trp?Ser?Ala?Glu?Phe?Asp?Val?Arg
580?????????????????585?????????????????590
gca?aac?ggc?ccc?caa?gat?ttt?gat?ccg?cag?cat?ttc?ctg?cag?gca?tat????1824
Ala?Asn?Gly?Pro?Gln?Asp?Phe?Asp?Pro?Gln?His?Phe?Leu?Gln?Ala?Tyr
595?????????????????600?????????????????605
caa?tcc?ggt?gtg?ttt?tcc?gag?gtt?ccc?att?cca?gca?cct?ggg?ata?aca????1872
Gln?Ser?Gly?Val?Phe?Ser?Glu?Val?Pro?Ile?Pro?Ala?Pro?Gly?Ile?Thr
610?????????????????615?????????????????620
gcc?aca?ttt?tgg?cac?ggg?aac?act?tta?gaa?gtg?cgc?act?gag?ctt?cgc????1920
Ala?Thr?Phe?Trp?His?Gly?Asn?Thr?Leu?Glu?Val?Arg?Thr?Glu?Leu?Arg
625?????????????????630?????????????????635?????????????????640
aca?gga?gct?att?ttt?gcc?ctg?ctc?aga?aca?ttg?ccc?gat?gcc?ctc?tgg????1968
Thr?Gly?Ala?Ile?Phe?Ala?Leu?Leu?Arg?Thr?Leu?Pro?Asp?Ala?Leu?Trp
645?????????????????650?????????????????655
atc?aac?gct?gtg?acc?cgc?ggt?gcg?acc?ctg?att?atc?cag?gca?gca?ctg????2016
Ile?Asn?Ala?Val?Thr?Arg?Gly?Ala?Thr?Leu?Ile?Ile?Gln?Ala?Ala?Leu
660?????????????????665?????????????????670
aag?ccc?ggc?ttc?gat?cga?gca?acg?gtg?gaa?cgc?tcc?gta?gtc?agg?tcg????2064
Lys?Pro?Gly?Phe?Asp?Arg?Ala?Thr?Val?Glu?Arg?Ser?Val?Val?Arg?Ser
675?????????????????680?????????????????685
ttg?gca?ggt?agc????????????????????????????????????????????????????2076
Leu?Ala?Gly?Ser
690
<210>??24
<211>??692
<212>??PRT
＜213〉brevibacterium
<400>??24
Met?Asn?Asn?Pro?Ala?Gln?Leu?Arg?Gln?Asp?Thr?Glu?Lys?Glu?Val?Leu
1???????????????5??????????????????10??????????????????15
Ala?Leu?Leu?Gly?Ser?Leu?Val?Leu?Pro?Ala?Gly?Thr?Ala?Leu?Ala?Ala
20??????????????????25??????????????????30
Thr?Gly?Ser?Leu?Ala?Arg?Ser?Glu?Leu?Thr?Pro?Tyr?Ser?Asp?Leu?Asp
35??????????????????40??????????????????45
Leu?Ile?Leu?Ile?His?Pro?Pro?Gly?Ala?Thr?Pro?Asp?Gly?Val?Glu?Asp
50??????????????????55??????????????????60
Leu?Trp?Tyr?Pro?Ile?Trp?Asp?Ala?Lys?Lys?Arg?Leu?Asp?Tyr?Ser?Val
65??????????????????70??????????????????75??????????????????80
Arg?Thr?Pro?Asp?Glu?Cys?Val?Ala?Met?Ile?Ser?Ala?Asp?Ser?Thr?Ala
85??????????????????90??????????????????95
Ala?Leu?Ala?Met?Leu?Asp?Leu?Arg?Phe?Ile?Ala?Gly?Asp?Glu?Asp?Leu
100?????????????????105?????????????????110
Cys?Ala?Lys?Thr?Arg?Arg?Arg?Ile?Val?Glu?Lys?Trp?Arg?Gln?Glu?Leu
115?????????????????120?????????????????125
Asn?Lys?Asn?Phe?Asp?Ala?Val?Val?Asp?Thr?Ala?Ile?Ala?Arg?Trp?Arg
130?????????????????135?????????????????140
Arg?Ser?Gly?Pro?Val?Val?Ala?Met?Thr?Arg?Pro?Asp?Leu?Lys?His?Gly
145?????????????????150?????????????????155?????????????????160
Arg?Gly?Gly?Leu?Arg?Asp?Phe?Glu?Leu?Ile?Lys?Ala?Leu?Ala?Leu?Gly
165?????????????????170?????????????????175
His?Leu?Cys?Asn?Val?Pro?Gln?Leu?Asp?Thr?Gln?His?Gln?Leu?Leu?Leu
180?????????????????185?????????????????190
Asp?Ala?Arg?Thr?Leu?Leu?His?Val?His?Ala?Arg?Arg?Ser?Arg?Asp?Val
195?????????????????200?????????????????205
Leu?Asp?Pro?Glu?Phe?Ala?Val?Asp?Val?Ala?Met?Asp?Leu?Gly?Phe?Val
210?????????????????215?????????????????220
Asp?Arg?Tyr?His?Leu?Gly?Arg?Glu?Ile?Ala?Asp?Ala?Ala?Arg?Ala?Ile
225?????????????????230?????????????????235?????????????????240
Asp?Asp?Gly?Leu?Thr?Thr?Ala?Leu?Ala?Thr?Ala?Arg?Gly?Ile?Leu?Pro
245?????????????????250?????????????????255
Arg?Arg?Thr?Gly?Phe?Ala?Phe?Arg?Asn?Ala?Ser?Arg?Arg?Pro?Leu?Asp
260?????????????????265?????????????????270
Leu?Asp?Val?Val?Asp?Ala?Asn?Gly?Thr?Ile?Glu?Leu?Ser?Lys?Lys?Pro
275?????????????????280?????????????????285
Asp?Leu?Asn?Asp?Pro?Ala?Leu?Pro?Leu?Arg?Val?Ala?Ala?Ala?Ala?Ala
290?????????????????295?????????????????300
Thr?Thr?Gly?Leu?Pro?Val?Ala?Glu?Ser?Thr?Trp?Ala?Arg?Leu?Asn?Glu
305?????????????????310?????????????????315?????????????????320
Cys?Pro?Pro?Leu?Pro?Glu?Pro?Trp?Pro?Ala?Asn?Ala?Ala?Gly?Asp?Phe
325?????????????????330?????????????????335
Phe?Arg?Ile?Leu?Ser?Ser?Pro?Lys?Asn?Ser?Arg?Arg?Val?Val?Lys?Asn
340?????????????????345?????????????????350
Met?Asp?Arg?His?Gly?Leu?Trp?Ser?Arg?Phe?Val?Pro?Glu?Trp?Asp?Arg
355?????????????????360?????????????????365
Ile?Lys?Gly?Leu?Met?Pro?Arg?Glu?Pro?Ser?His?Ile?Ser?Thr?Ile?Asp
370?????????????????375?????????????????380
Glu?His?Ser?Leu?Asn?Thr?Val?Ala?Gly?Cys?Ala?Leu?Glu?Thr?Val?Thr
385?????????????????390?????????????????395?????????????????400
Val?Ala?Arg?Pro?Asp?Leu?Leu?Val?Leu?Gly?Ala?Leu?Tyr?His?Asp?Ile
405?????????????????410?????????????????415
Gly?Lys?Gly?Phe?Pro?Arg?Pro?His?Glu?Gln?Val?Gly?Ala?Glu?Met?Val
420?????????????????425?????????????????430
Ala?Arg?Ala?Ala?Ser?Arg?Met?Gly?Leu?Asn?Leu?Arg?Asp?Arg?Ala?Ser
435?????????????????440?????????????????445
Val?Gln?Thr?Leu?Val?Ala?Glu?His?Thr?Ala?Val?Ala?Lys?Ile?Ala?Ala
450?????????????????455?????????????????460
Arg?Leu?Asp?Pro?Ser?Ser?Glu?Gly?Ala?Val?Asp?Lys?Leu?Leu?Asp?Ala
465?????????????????470?????????????????475?????????????????480
Val?Arg?Tyr?Asp?Leu?Val?Thr?Leu?Asn?Leu?Leu?Glu?Val?Leu?Thr?Glu
485?????????????????490?????????????????495
Ala?Asp?Ala?Lys?Ala?Thr?Gly?Pro?Gly?Val?Trp?Thr?Ala?Arg?Leu?Glu
500?????????????????505?????????????????5l0
His?Ala?Leu?Arg?Ile?Val?Cys?Lys?Arg?Ala?Arg?Asp?Arg?Leu?Thr?Asp
515?????????????????520?????????????????525
Ile?Arg?Pro?Val?Ala?Pro?Met?Ile?Ala?Pro?Arg?Ser?Glu?Ile?Gly?Leu
530?????????????????535?????????????????540
Val?Glu?Arg?Asp?Gly?Val?Phe?Thr?Val?Gln?Trp?His?Gly?Glu?Asp?Leu
545?????????????????550?????????????????555?????????????????560
His?Arg?Ile?Leu?Gly?Val?Ile?Tyr?Ala?Lys?Gly?Trp?Thr?Ile?Thr?Ala
565?????????????????570?????????????????575
Ala?Arg?Met?Leu?Ala?Asn?Gly?Gln?Trp?Ser?Ala?Glu?Phe?Asp?Val?Arg
580?????????????????585?????????????????590
Ala?Asn?Gly?Pro?Gln?Asp?Phe?Asp?Pro?Gln?His?Phe?Leu?Gln?Ala?Tyr
595?????????????????600?????????????????605
Gln?Ser?Gly?Val?Phe?Ser?Glu?Val?Pro?Ile?Pro?Ala?Pro?Gly?Ile?Thr
610?????????????????615?????????????????620
Ala?Thr?Phe?Trp?His?Gly?Asn?Thr?Leu?Glu?Val?Arg?Thr?Glu?Leu?Arg
625?????????????????630?????????????????635?????????????????640
Thr?Gly?Ala?Ile?Phe?Ala?Leu?Leu?Arg?Thr?Leu?Pro?Asp?Ala?Leu?Trp
645?????????????????650?????????????????655
Ile?Asn?Ala?Val?Thr?Arg?Gly?Ala?Thr?Leu?Ile?Ile?Gln?Ala?Ala?Leu
660?????????????????665?????????????????670
Lys?Pro?Gly?Phe?Asp?Arg?Ala?Thr?Val?Glu?Arg?Ser?Val?Val?Arg?Ser
675?????????????????680?????????????????685
Leu?Ala?Gly?Ser
690
Claims (9)
1, a kind of coryneform bacteria with production L-arginine or L-Methionin ability, this coryneform bacteria is improved the activity of its glutamine synthetase through modifying.
2, the described coryneform bacteria of claim 1, this coryneform bacteria makes it be reduced or eliminate by the active regulating effect of adenylylation to glutamine synthetase through modifying.
3, the described coryneform bacteria of claim 2 wherein makes it be reduced or eliminate by the active regulating effect of adenylylation to glutamine synthetase by following one or more features:
A) bacterium comprises glutamine synthetase, and the activity that adenylylation carries out passed through of described enzyme is regulated and is eliminated,
B) activity is lowered in the born of the same parents of Glutamine-synthetase adenylyltransferase,
C) activity is lowered in the proteic born of the same parents of PII, and
D) nitrogen metabolism regulate that protein is modified so that the born of the same parents of glutamine synthetase in increased activity.
4, the described coryneform bacteria of claim 3, the gene of coding Glutamine-synthetase adenylyltransferase is destroyed on the wherein said bacterial chromosome.
5, the described coryneform bacteria of claim 3, wherein said ammonia metabolism are regulated the product that albumen is the amtR gene of the effect of can not bringing into normal play.
6, the described coryneform bacteria of claim 5, the amtR gene on the wherein said bacterial chromosome is destroyed.
7, each described coryneform bacteria of claim 1-6, this coryneform bacteria be through modifying, and makes the effect of can not bringing into normal play of arginine repressor.
8, the described coryneform bacteria of claim 7, the gene of coding arginine repressor is destroyed on the wherein said bacterial chromosome.
9, a kind of method of producing L-arginine or L-Methionin comprises the following step:
A) in substratum, cultivate among the claim 1-8 each described coryneform bacteria and
B) in substratum, gather L-arginine or L-Methionin, and
C) collect L-arginine or L-Methionin from substratum.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003056129 | 2003-03-03 | ||
| JP56129/03 | 2003-03-03 | ||
| JP56129/2003 | 2003-03-03 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1550546A true CN1550546A (en) | 2004-12-01 |
| CN100366731C CN100366731C (en) | 2008-02-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100330506A Expired - Fee Related CN100366731C (en) | 2003-03-03 | 2004-03-03 | Method for producing L-arginine or L-lysine by fermentation |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US20050014236A1 (en) |
| EP (1) | EP1460128B1 (en) |
| KR (1) | KR101086205B1 (en) |
| CN (1) | CN100366731C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109370975A (en) * | 2018-12-05 | 2019-02-22 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield |
| CN109943548A (en) * | 2019-04-03 | 2019-06-28 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield |
| CN110734887A (en) * | 2018-07-18 | 2020-01-31 | 中国科学院微生物研究所 | Genetically engineered bacterium for producing N-acetylglutamic acid and construction method and application thereof |
Families Citing this family (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL341895A1 (en) * | 1999-08-12 | 2001-02-26 | Ajinomoto Kk | Plasmide autonomously replicable in corynebacter bacteria |
| EP1424397B1 (en) | 2002-11-26 | 2011-01-19 | Ajinomoto Co., Inc. | Method for producing L-glutamine and L-glutamine producing bacterium |
| JP4380305B2 (en) * | 2003-11-21 | 2009-12-09 | 味の素株式会社 | Method for producing L-amino acid by fermentation |
| US8211688B2 (en) | 2004-06-25 | 2012-07-03 | Kyowa Hakko Bio Co., Ltd. | Process for producing L-glutamine using Escherichia coli with deficient glnB and glnE function |
| US7205132B2 (en) * | 2004-09-10 | 2007-04-17 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
| JP4595506B2 (en) * | 2004-11-25 | 2010-12-08 | 味の素株式会社 | L-amino acid-producing bacterium and method for producing L-amino acid |
| US7794989B2 (en) | 2004-12-28 | 2010-09-14 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
| US20070004014A1 (en) * | 2005-06-29 | 2007-01-04 | Yuichiro Tsuji | Method for producing l-threonine |
| KR100854234B1 (en) | 2006-07-13 | 2008-08-25 | 씨제이제일제당 (주) | A nucleotide sequence of a mutant argF with increased activity and a method for producing L-arginine using a transformed cell containing the same |
| JP2010041920A (en) | 2006-12-19 | 2010-02-25 | Ajinomoto Co Inc | Method for producing l-amino acid |
| JP2010130899A (en) | 2007-03-14 | 2010-06-17 | Ajinomoto Co Inc | Microorganism producing l-glutamic acid-based amino acid, and method for producing amino acid |
| DE102008001874A1 (en) * | 2008-05-20 | 2009-11-26 | Evonik Degussa Gmbh | Process for the preparation of L-amino acids |
| BR112013016373B1 (en) | 2011-11-11 | 2021-05-18 | Ajinomoto Co., Inc | method for producing a target substance |
| JP2016165225A (en) | 2013-07-09 | 2016-09-15 | 味の素株式会社 | Method for producing useful substance |
| JP2016192903A (en) | 2013-09-17 | 2016-11-17 | 味の素株式会社 | Method for manufacturing l-amino acid from biomass derived from seaweed |
| EP3061828A4 (en) | 2013-10-23 | 2017-03-15 | Ajinomoto Co., Inc. | Method for producing target substance |
| JP6623690B2 (en) | 2015-10-30 | 2019-12-25 | 味の素株式会社 | Method for producing glutamic acid-based L-amino acid |
| JP7066977B2 (en) | 2017-04-03 | 2022-05-16 | 味の素株式会社 | Manufacturing method of L-amino acid |
| WO2020071538A1 (en) | 2018-10-05 | 2020-04-09 | Ajinomoto Co., Inc. | Method for producing target substance by bacterial fermentation |
| CN111172186B (en) * | 2020-01-16 | 2023-04-07 | 天津科技大学 | Steroid prodrug production method capable of reducing nitrogen source dosage |
| KR102198072B1 (en) * | 2020-03-04 | 2021-01-04 | 씨제이제일제당 주식회사 | A modified polypeptide of glutamine synthetase and a method for L-glutamine using the same |
| WO2022092018A1 (en) | 2020-10-28 | 2022-05-05 | 味の素株式会社 | Method of producing l-amino acid |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5610036B1 (en) * | 1969-07-23 | 1981-03-05 | ||
| PL341895A1 (en) * | 1999-08-12 | 2001-02-26 | Ajinomoto Kk | Plasmide autonomously replicable in corynebacter bacteria |
| US7160705B2 (en) * | 2000-04-28 | 2007-01-09 | Ajinomoto Co., Inc. | Arginine repressor deficient strain of coryneform bacterium and method for producing L-arginine |
| JP2002051790A (en) * | 2000-04-28 | 2002-02-19 | Ajinomoto Co Inc | Arginine repressor-defected strain of coryne form bacterium and method for producing l-arginine |
| JP4560998B2 (en) * | 2001-02-05 | 2010-10-13 | 味の素株式会社 | Method for producing L-glutamine by fermentation and L-glutamine producing bacteria |
| EP1424397B1 (en) * | 2002-11-26 | 2011-01-19 | Ajinomoto Co., Inc. | Method for producing L-glutamine and L-glutamine producing bacterium |
-
2004
- 2004-03-02 EP EP04004888.6A patent/EP1460128B1/en not_active Expired - Fee Related
- 2004-03-02 US US10/790,224 patent/US20050014236A1/en not_active Abandoned
- 2004-03-03 KR KR1020040014386A patent/KR101086205B1/en active IP Right Grant
- 2004-03-03 CN CNB2004100330506A patent/CN100366731C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734887A (en) * | 2018-07-18 | 2020-01-31 | 中国科学院微生物研究所 | Genetically engineered bacterium for producing N-acetylglutamic acid and construction method and application thereof |
| CN110734887B (en) * | 2018-07-18 | 2021-09-03 | 中国科学院微生物研究所 | Genetically engineered bacterium for producing N-acetylglutamic acid and construction method and application thereof |
| CN109370975A (en) * | 2018-12-05 | 2019-02-22 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield |
| CN109943548A (en) * | 2019-04-03 | 2019-06-28 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield |
Also Published As
| Publication number | Publication date |
|---|---|
| US20050014236A1 (en) | 2005-01-20 |
| KR101086205B1 (en) | 2011-11-24 |
| EP1460128B1 (en) | 2016-11-30 |
| EP1460128A1 (en) | 2004-09-22 |
| KR20040078588A (en) | 2004-09-10 |
| CN100366731C (en) | 2008-02-06 |
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